Hla-restricted, peptide-specific binding proteins

ABSTRACT

Antigen binding proteins with TCR-like paratopes, that is, with an antigen binding region specific for an HLA-A2 restricted peptide are disclosed. The antigen binding proteins encompass antibodies in a variety of forms, including full-length antibodies, substantially intact antibodies, Fab fragments, F(ab′)2 fragments, and single chain Fv fragments. Fusion proteins, such as scFv fusions with immunoglobulin or T-cell receptor domains, incorporating the specificity of the antigen binding region for each peptide are also contemplated by the invention. Furthermore, immunoconjugates may include antibodies to which is linked a radioisotope, fluorescent or other detectable marker, cytotoxin, or other molecule are also encompassed by the invention. Among other things, immunoconjugates can be used for delivery of an agent to elicit a therapeutic effect or to facilitate an immune effector function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.61/463,082, filed Feb. 11, 2011, entitled GENERATION AND USE OF HLA-A2RESTRICTED, PEPTIDE-SPECIFIC MONOCLONAL ANTIBODIES AND CHIMERIC ANTIGENRECEPTORS. This Provisional Application is hereby incorporated byreference in its entirety into the present disclosure.

SEQUENCE LISTING

The instant application contains a Sequence Listing, created on Feb. 2,2012; the file, in ASCII format, is designated3314019AWO_seqlisting_ST25.txt and is 47.8 kilobytes in size. The fileis hereby incorporated by reference in its entirety into the instantapplication.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates generally to antigen-binding proteinmolecules involved in immune function. More particularly, the presentinvention relates to recombinant antibodies, chimeric antigen receptorsand fragments thereof with specificity for an HLA-restricted peptide,where the peptide is derived from a cellular or viral protein ofinterest.

2. Background Information

Advances in adoptive T cell immunotherapy have led to several promisingoptions for cancer patients in the past decade. T-cell basedimmunotherapy for cancer stemmed from studies which showed a correlationof increased numbers of tumor infiltrating lymphocytes (TILs) insurgical specimens and patient outcome. It is generally believed thatthis infiltration of TILs represents activation of an anti-tumormechanism and that the infiltration was mediated through the expressionof tumor associated antigens in the context of MHC. These findingseventually led researchers to try and take advantage of antigen-specificT cells for the treatment of cancer.

For induction of cytotoxic T-cell (CTL) responses, intracellularproteins are usually degraded by the proteasome or endo/lysosomes, andthe resulting peptide fragments bind to MHC class I or II molecules.These peptide-MHC complexes are displayed at the cell surface where theyprovide targets for T cell recognition via a peptide-MHC (μMHC)-T cellreceptor (TCR) interaction. Vaccinations with peptides derived fromcellular and viral protein can induce HLA-A0201-restricted cytotoxic CD8T cells, which are capable of killing tumor cells or virally-infectedcells.

Antibodies are increasingly being used as therapeutic agents to fightcancer, autoimmune disease and infection. Therapeutic antibodies havebeen exploited based on their multiple mechanisms of action, whichinclude the following: 1) naked antibodies killing tumor cells directlyby ADCC or CDC (e.g. trastuzumab), 2) blocking or stimulating a cellmembrane molecule to induce cell death (e.g. cetuximab), 3) neutralizinga secreted moiety (e.g. bevacizumab), 4) killing via an attached moietysuch as a drug, toxin, radioisotope and 5) modulating the immune systemvia T cell effector functions.

In almost all cases, to generate a therapeutic benefit, antibodies haveto possess critical properties including high affinity for theirtargeted antigen, minimal acute and long-term side effects, and inspecific applications, high affinity for human Fc receptors (4). Inaddition, the targeted antigen has to be expressed at high levels ontumors but not on normal tissues (specificity or selectivity),consistently expressed in the specific tumor among patients and withinpatients (low heterogeneity), and should either be essential for thesurvival of the cancer cell or unlikely to be down regulated.

To achieve these attributes, researchers can now reengineer existingantibodies to make them less immunogenic, modifying both protein andcarbohydrate residues in the Fc regions to enhance ADCC and CDC,shrinking their sizes for potentially better tumor penetration, mutatingthe variable regions to improve affinity, increasing avidity by changingantibody valency, and constructing novel antibody-fusion proteins suchas those for multi-step targeting (5) and for redirecting immune cellsby way of a chimeric antigen receptor (CAR). Furthermore, researcherscontinue to define the structural attributes and the hostcharacteristics responsible for success among currently approvedantibodies (6).

With the objective of eliminating or neutralizing the pathogenic agentor disease target, including bacterial, viral or tumor targets,antigen-specific, antibody-based treatments are particularly attractivebecause of the antibody's exquisite specificity.

SUMMARY OF THE INVENTION

The present invention, therefore, is based on the identification ofantigen-specific binding sequences from which a variety ofantigen-binding proteins can be produced, for example, an antibodyspecific for an antigen that represents a complex of a protein fragment(peptide) and an HLA molecule similar to that typically recognized by aT-cell receptor following antigen processing and presentation of theprotein to the T-cell. Phage display is used to select an initialantigen-binding molecule that can be used to engineer theantigen-binding proteins of the invention, which include antibodies andchimeric antigen receptors (CARs).

In one aspect, therefore, the invention relates to an isolatedantigen-binding protein or antigen-binding fragment thereof comprisingone of:

-   -   (A) an antigen binding region having the amino acid sequence of        one of SEQ ID NOS: 2, 5, 8, 10, 13, 14, 17, 20;    -   (B) an antigen binding region comprising a V_(H) and V_(L),        respectively, with amino acid sequences selected from SEQ ID        NOs: 22 and 23; 24 and 25; 26 and 27; 28 and 29; 30 and 31; 32        and 33; 34 and 35; and 36 and 37; or    -   (C) (i) the following three light chain (LC) complementarity        determining regions (CDRs):    -   (a) a LC CDR1 comprising the amino acid sequence of SEQ ID NO:        56; and    -   (b) a LC CDR2 and CDR3 comprising respectively, the amino acid        sequence of SEQ ID NOs: 57 and 64, 58 and 65, 59 and 66, 60 and        67, 61 and 68, 61 and 69, 62 and 70 and 63 and 71; and    -   (ii) the following three heavy chain (HC) CDRs:    -   (a) a HC CDR1 comprising the amino acid sequence of SEQ ID NO:        38; and    -   (b) a LC CDR2 and CDR3 comprising respectively the amino acid        sequence of one of SEQ ID NOs: 40 and 48, 41 and 49, 42 and 50,        43 and 51, 44 and 52, 45 and 53, 46 and 54 and 47 and 55.

In a related aspect, the invention relates to an isolatedantigen-binding protein or antigen-binding fragment thereof, wherein theisolated antigen-binding protein is an antibody or a chimeric antigenreceptor. The antibody is a full-length antibody, a substantially intactantibody, a Fab fragment, a F(ab′)₂ fragment or a single chain variablefragment (scFv).

In the isolated antigen-binding protein, whether an antibody or CAR, theantigen-binding region specifically binds to an epitope of anHLA-peptide complex.

Peptides that are recognized by the antigen-binding proteins of theinvention as part of an HLA-peptide complex include, but are not limitedto, a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO:1); apeptide with the amino acid sequence LLDFVRFMGV (SEQ ID NO:4); a peptidewith the amino acid sequence RLTRFLSRV (SEQ ID NO: 7); a peptide withthe amino acid sequence RIITSTILV (SEQ ID NO: 12); and a peptide withthe amino acid sequence LLEEMFLTV (SEQ ID NO:19). In some embodiments,the peptide is recognized in associate with an HLA-A2 antigen.

In yet another aspect, the isolated antigen-binding protein of theinvention is a scFv comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 2, 5, 8, 10, 13, 14, 17 and 20.

In a related aspect, the isolated antigen-binding protein is a fusionprotein comprising an antigen-binding region as disclosed in any ofTables 1-8.

In another aspect, the invention relates to an immunoconjugatecomprising a first component which is an antigen-binding protein, orantigen-binding fragment thereof as disclosed herein. Theimmunoconjugate comprises a second component that is a cytotoxin, adetectable label, a radioisotope, a therapeutic agent, a binding proteinor a molecule having a second amino acid sequence. Where the secondcomponent is a binding protein or second antibody, the binding proteinor second antibody has binding specificity for a target that isdifferent from the HLA-peptide complex.

In a related aspect, therefore, the present invention relates tobispecific antibody comprising an antigen-binding protein or functionalfragment thereof as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of bacterial supernatant from individual EBNA3CscFv clones 315, 335, 327 and 345 (A) and purified EBNA clone 315 scFv(B) to various HLA-A2-peptide complexes demonstrating that clone 315 ishighly specific for the HLA-A2-LLDFVRFMGV complex.

FIG. 2 the binding of bacterial supernatant from individual WT-1 scFvclones 42, 43 and 45 (A) and purified WT-1 clone 45 scFv (B) to variousHLA-A2-peptide complexes demonstrating that WT-1 clones 42, 43 and 45are highly specific for the recombinant HLA-A2-RMFPNAPYL complex.

FIG. 3 shows that HLA-A2 can be detected on TAP-deficient (TAE) T2 cellsthat were either pulsed or unpulsed with LLDFVRFMGV or another(irrelevant) peptide (A) but that EBNA clone 315 scFv recognizes T2cells that have been pulsed with LLDFVRFMGV but not unpulsed cells orcells pulsed with irrelevant peptide (B) with a lower limit of detectionat about 78 nM (C).

FIG. 4 shows that HLA-A2 can be detected on TAP-deficient (TAP⁻) T2cells that were either pulsed or unpulsed with RMFPNAPYL or LLDFVRFMGV(A) but that WT-1 clone 45 scFv recognizes T2 cells that have beenpulsed with RMFPNAPYL but not unpulsed cells or cells pulsed withLLDFVRFMGV (B)

FIG. 5 shows that when DIMT (A) and 6268A (B) BLCLs are incubated withLLDFVRFMGV (middle panel) or KLQCVDLHV peptides (right panel) andstained with EBNA clone 315 scFv, only HLA-A2⁺ DIMT peptide-pulsed withLLDFVRFMGV could be stained, showing that EBNA clone 315 and LLDFVRFMGVare HLA-A2 restricted; a time course (bottom panel) shows that theHLA-A2-LLDFVRFMGV complex is stable on the cell surface.

FIG. 6 shows the Tomlinson library vector used in PCR to add appropriaterestriction enzyme sites to either side of the WT1 Clone 45 and EBNAClone 315 scFv sequences (FIG. 6A). FIG. 6B shows the digested PCRproducts as they appeared on a 1% agarose gel following digestion withNheI and ApaI.

FIG. 7 shows the full IgG expression vector (FIG. 7A) that was used togenerate an expression vector for scFv-Fc fusion proteins (FIG. 7B). A.The structure of the proprietary IgG expression vector (11381 bp). Thevector expresses the heavy and light chains under two separate CMVpromoters. The variable heavy chain (V_(H)) is fused to the first,second and third constant heavy chains (CH_(1, 2, 3)) and expressedunder one promoter while the variable light chain (V_(L)) is fused tothe constant light chain (C_(L)) and expressed under a differentpromoter. This vector was further modified to lack the first constantregion of the heavy chain (CH₁), and this vector was used for theconstruction of scFv-Fc fusion proteins. B. After excision of the V_(H)from the IgG vector using NheI and ApaI, the pre-digested, purified scFvPCR products were ligated to the IgG vector to allow for the expressionof the scFv fused to the CH_(2, 3) domains (Fc).

FIG. 8 shows the results of binding studies using EBNA Clone 315 scFv-Fcin which purified EBNA Clone 315 scFv-Fc was shown to maintain itsbinding ability towards the recombinant complex when tested for bindingon an ELISA plate coated with or without HLA-A2-LLDFVRFMGV (FIG. 8A) andwhen tested for binding on T2 cells pulsed with or without theLLDFVRFMGV peptide (FIG. 8B). When T2 cells were incubated withdecreasing concentrations of the LLDFVRFMGV peptide and subsequentlystained with EBNA Clone 315 scFv-Fc, a lower limit of detection wasdemonstrated to be in the same range as the scFv (200 nM-20 nM) (FIG.8C).

FIG. 9 shows the results of kinetics determination of EBNA Clone 315 toHLA-A2-LLDFVRFMGV using surface plasmon resonance.

FIG. 10 shows the results of HLA-A2-LLDFVRFMGV complex quantitation onT2 cells using fluorescently-conjugated EBNA Clone 315 scFv-Fc (FIG.10A). Fluorescently-conjugated EBNA Clone 315 scFv-Fc was tested forbinding on T2 cells pulsed with (20, 10, or 5 μM) or without (0 μM) theLLDFVRFMGV peptide in serum-free IMDM media at 37° C. for 5 hours. Thecells, in addition to beads containing known amounts of anti-human IgG₁antibodies, were stained with the scFv-Fc and the cell's fluorescenceintensity was correlated to that of the beads and their number ofbinding sites. Using these four peptide concentrations and correspondingnumber of complexes, a standard curve was created with an R² value of0.9948. Figure B shows a close-up view of the lower end of the peptideand complex spectrum.

FIG. 11 shows the results of binding and specificity studies whenpurified WT1 Clone 45 scFv-Fc was tested for binding on an ELISA platecoated with or without HLA-A2-RMFPNAPYL (FIG. 11A). FIG. 11B shows thatwhen purified WT1 Clone 45 scFv-Fc was tested for binding on T2 cellspulsed with the RMFPNAPYL or RLTRFLSRV peptide (40 μM), the scFv-Fc(unfilled lines) was only able to recognize RMFPNAPYL-pulsed T2 cells.

FIG. 12 shows that when DIMT (top) and 6268A (bottom) BLCLs wereincubated with RMFPNAPYL (right panel) and the peptide-pulsed BLCLs werestained with WT1 Clone 315 scFv-Fc (unfilled lines) or a control scFv(filled lines), only the HLA-A2-positive RMFPNAPYL peptide-pulsed DIMTBLCLs could be stained.

FIG. 13 shows EBNA Clone 315 scFv-Fc mediated ADCC (measured using ⁵¹Crrelease) of LLDFVRFMGV peptide-pulsed cells.

FIG. 14 shows the MSCV-based vector (left panel) containing an IRES-GFPsequence along with ampicilin-resistance used for transduction andexpression of anti-EBNA CAR in NK92MI cells. A The EBNA Clone 315 scFvsequence was cloned into the CAR gene (EBNA CAR) and further cloned intoan MSCV-based vector (left panel) which contained an IRES-GFP sequencealong with ampicilin-resistance. The resulting CAR (right panel) iscomposed of the scFv and hinge region on the extracellular surface, atransmembrane domain, along with 4-1BB and the CD3ζ chain present withinthe cell. B After retroviral packaging using 293T GP2 cells andtransduction into NK92MI cells, approximately 24% of the NK92MI cellscontained the construct based on GFP expression (left panel; unfilledlines) when compared to mock transduced (empty retrovirus) NK92MI cells(left panel; filled lines). Of the GFP-positive cells, the top 20% wereflow cytometrically sorted and expanded to yield a population of stablytransduced cells which were greater than 90% GFP positive (right panel).Retroviral transduction was done on three separate occasions, with 24%being the highest efficiency.

FIG. 15 shows EcoRI and XhoI digestion validation of the WT1 Clone 45CAR vector. A. Along with sequence validation, plasmids isolated from 8different bacterial colonies, after ligation, transformation and EcoRIand XhoI digestion, were run on a 1% agarose gel. Based on the lambdaHindIII and 100 bp markers, it was determined that the bands were thecorrect size (˜1500 bp and ˜6000 bp). B. The structure of the resultingWT1 Clone 45 CAR vector has the same components as the original St. JudeCAR vector with the only difference being the scFv sequence.

FIG. 16 shows that Clone 315 CAR-expressing NK92MI cells canspecifically detect the HLA-A2-EBNA3C complex on peptide-pulsed T2 cellsvia CD107a expression. T2 cells were pulsed with or without LLDFVRFMGVor YMFPNAPYL peptides at 20 μM. CAR-equipped NK92MI cells were thencultured in media containing an anti-CD107a-PE conjugated antibody at37° C. for 5 hours with or without peptide pulsed or unpulsed cells. A.CAR-equipped NK92MI cells were gated based on GFP fluorescence andanalyzed for CD107a expression. NK92MI cells which were cultured withoutany T2 cells or those which were cocultured with unpulsed andYMFPNAPYL-pulsed T2 cells were unreactive while NK92MI cells which werecocultured with LLDFVRFMGV-pulsed T2 cells led to a 27% increase inCD107a expression above background levels. B. T2 cells were pulsed withdecreasing concentrations of LLDFVRFMGV and subsequently cocultured withCAR-equipped NK92MI cells. NK92MI cells presented noticeable amounts ofCD107a on their cell surface even when T2 cells were pulsed with only 10nM of peptide.

FIG. 17 shows the results of flow cytometry in which HLA-A2⁺ (DIMT) andHLA-A2⁻ (6268A) BLCLs were pulsed with LLDFVRFMGV and CAR-equippedNK92MI cells were then cultured in media containing an anti-CD107a-PEconjugated antibody with or without peptide pulsed or unpulsed cells.CAR-equipped NK92MI cells were gated based on GFP fluorescence andanalyzed for CD107a expression. NK92MI cells which were cultured withoutany BLCL or those which were cocultured with LLDFVRFMGV-pulsed 6268ABLCL were unreactive while NK92MI cells which were cocultured withunpulsed DIMT BLCL or LLDFVRFMGV-pulsed DIMT BLCL led to a 0.5% and 25%increase in CD107a expression above background levels (pulsed 6268A)showing that EBNA Clone 315 CAR-expressing NK92MI cells can specificallydetect the HLA-A2-EBNA3C complex on peptide-pulsed BLCLs via CD107aexpression.

FIG. 18 shows the results of a ⁵¹Cr release assay in which T2 cells werepulsed with or without decreasing concentrations of LLDFVRFMGV.CAR-equipped NK92MI cells were cocultured with ⁵¹Cr-labeled T2 cells ata 3:1 E:T ratio demonstrating that EBNA Clone 315 CAR-expressing NK92MIcells can specifically detect the HLA-A2-EBNA3C complex onpeptide-pulsed T2 cells. Even with 2 nM of peptide, peptide-specificcytotoxicity could be observed when compared to unpulsed T2 cells.

FIG. 19 shows EBNA Clone 315 CAR-expressing NK92MI cells canspecifically detect the HLA-A2-EBNA3C complex on peptide-pulsed BLCLsvia ⁵¹Cr release. BLCLs were pulsed with LLDFVRFMGV. CAR-equipped NK92MIcells were then cultured with ⁵¹Cr labeled target cells. A. CAR equippedNK92MI cells were able to specifically differentiate between peptidepulsed DIMT and 6268A BLCL, with a clear difference in cytotoxicitybetween the two different targets. B. CAR-mediated killing ofpeptide-pulsed DIMT BLCL could be blocked using the EBNA Clone 315scFv-Fc fusion protein, but not by an irrelevant, isotype-matchedscFv-Fc, at a 20:1 E:T ratio.

FIG. 20 shows the results of a ⁵¹Cr release assay in which CAR-equippedNK92MI cells were cultured with ⁵¹Cr labeled, unpulsed BLCLs. A.CAR-equipped NK92MI cells were more reactive towards the HLA-A2⁺ DIMTand JG19 BLCL versus the HLA-A2⁻ 6268A and GKO BLCL when cocultured inthe absence of any exogenous peptide. B. CAR-mediated killing ofunpulsed DIMT BLCL could be blocked using the EBNA Clone 315 scFv-Fcfusion protein but not by an irrelevant, isotype-matched scFv-Fc, at a10:1 E:T ratio demonstrating that EBNA Clone 315 CAR-expressing NK92MIcells can specifically detect the HLA-A2-EBNA3C complex on HLA-A2⁺BLCLs.

FIG. 21 shows the results of a ⁵¹Cr release assay of EBNA in whichCD16(V)-expressing NK92MI cells were cultured with ⁵¹Cr labeled,LLDFVRFMGV-pulsed DIMT BLCL and either EBNA Clone 315 or an irrelevantscFv-Fc. At an E:T ratio of 15:1, EBNA Clone 315 scFv-Fc was able tokill 30-35% of target cells.

FIG. 22 shows the results of a ⁵¹Cr release assay of EBNA in which Clone315 CAR-expressing NK92MI cells were cultured with LLDFVRFMGV-pulsedDIMT BLCL as above and either EBNA Clone 315 or an irrelevant scFv-Fc.At the same E:T ratio as in FIG. 21, the CAR-equipped cells were able tokill 80-90% of target cells, with specific inhibition using EBNA ClonescFv-Fc demonstrating that CAR-mediated killing is more potent thanscFv-Fc-mediated ADCC on peptide-pulsed DIMT BLCL.

FIG. 23 shows the MSCV-based vector (left panel) containing an IRES-GFPsequence along with ampicilin-resistance used for transduction andexpression of anti-WT1 CAR in NK92MI cells. A. The WT1 Clone 45 scFvsequence was cloned into the CAR gene (anti-WT1 CAR) and further clonedinto the MSCV-based vector. The resulting CAR (right panel) is composedof the scFv and hinge region on the extracellular surface, atransmembrane domain, along with 4-1BB and the CD3ζ chain present withinthe cell. B After retroviral packaging using 293T GP2 cells andtransduction into NK92MI cells, approximately 27.5% of the NK92MI cellscontained the construct based on GFP expression (left panel; unfilledlines) when compared to mock transduced (empty retrovirus) NK92MI cells(left panel; filled lines). Of the GFP-positive cells, the top 20% wereflow cytometrically sorted and expanded to yield a population of stablytransduced cells which were greater than 98% GFP positive (right panel).

FIG. 24 shows the results of a ⁵¹Cr release assay in which CAR-equippedNK92MI cells were able to specifically differentiate between peptidepulsed DIMT (▪) and 6268A BLCL (), with a clear difference incytotoxicity between the two different targets demonstrating that NK92MIcells expressing WT1 Clone 45 CAR can specifically detect theHLA-A2-RMFPNAPYL complex on peptide-pulsed BLCLs.

FIG. 25 shows that CAR-mediated killing of peptide-pulsed DIMT BLCLcould be blocked using a commercial anti-HLA-A2 antibody (5 μg/ml), butnot by an irrelevant, isotype-matched antibody (5 μg/ml), at a 9:1 E:Tratio.

FIG. 26 shows that NK92MI cells expressing WT1 Clone 45 CAR canspecifically detect the HLA-A2-RMFPNAPYL complex on DIMT BLCL via ⁵¹Crrelease. A. CAR equipped NK92MI cells were able to specificallydifferentiate between DIMT and 6268A BLCL, with a clear difference incytotoxicity between the two different targets. B. CAR-mediated killingof DIMT BLCL could be blocked using the WT1 Clone 45 scFv-Fc fusionprotein (20 μg/ml), but not by an irrelevant, isotype-matched scFv-Fc,at a 2:1 E:T ratio.

FIG. 27 shows that NK92MI cells expressing WT1 Clone 45 CAR canspecifically detect the HLA-A2-RMFPNAPYL complex on peptide-pulsed BLCLsvia ⁵¹Cr release. A CAR-equipped NK92MI cells were able to specificallydifferentiate between peptide pulsed DIMT and 6268A BLCL, with a cleardifference in cytotoxicity between the two different targets.

FIG. 28 shows that NK92MI cells expressing WT1 Clone 45 CAR canspecifically detect the HLA-A2-RMFPNAPYL complex on 697 and OVCAR-3cells via ⁵¹Cr release.

DETAILED DESCRIPTION OF THE INVENTION

All patents, publications, applications and other references citedherein are hereby incorporated in their entirety into the presentapplication.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, cell biology, biochemistry, andimmunology are used, which are within the skill of the art. Thesetechniques are described in greater detail in, for example, MolecularCloning: a Laboratory Manual 3^(rd) edition, J. F. Sambrook and D. W.Russell, ed. Cold Spring Harbor Laboratory Press 2001; RecombinantAntibodies for Immunotherapy, Melvyn Little, ed. Cambridge UniversityPress 2009; “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M.Ausubel et al., eds., 1987, and periodic updates); “PCR: The PolymeraseChain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide toMolecular Cloning” (Perbal Bernard V., 1988); “Phage Display: ALaboratory Manual” (Barbas et al., 2001). The contents of thesereferences and other references containing standard protocols, widelyknown to and relied upon by those of skill in the art, includingmanufacturers' instructions are hereby incorporated by reference as partof the present disclosure.

The following abbreviations are used throughout the application:

ADCC: Antibody-dependent cellular cytotoxicity

ALL: Acute lymphocytic leukemia

AML: Acute myeloid leukemia

APC: Antigen presenting cell

β2M: Beta-2-microglobulin

BiTE: Bi-specific T cell engaging antibody

BLCL: EBV-transformed B-cell lymphoblastic cell line

CAR: Chimeric antigen receptor

CDC: Complement dependent cytotoxicity

CMC: Complement mediated cytotoxicity

CDR: Complementarity determining region (see also HVR below)

C_(L): Constant domain of the light chain

CH₁: 1^(st) constant domain of the heavy chain

CH_(1, 2, 3): 1^(st), 2^(nd) and 3^(rd) constant domains of the heavychain

CH_(2, 3): 2^(nd) and 3^(rd) constant domains of the heavy chain

CHO: Chinese hamster ovary

CTL: Cytotoxic T cell

EBNA3C: Epstein-Barr nuclear antigen 3C

EBV: Epstein-Barr virus

ECMV: Encephalomyocarditis virus

ER: Endoplasmic reticulum

E:T Ratio: Effector:Target ratio

Fab: Antibody binding fragment

FACS: Flow assisted cytometric cell sorting

FBS: Fetal bovine serum

GFP: Green fluorescence protein

HC: Heavy chain

HEL: Hen egg lysozyme

HLA: Human leukocyte antigen

HVR-H: Hypervariable region-heavy chain (see also CDR)

HVR-L: Hypervariable region-light chain

Ig: Immunoglobulin

IPTG: isopropyl-1-thio-β-D-galactopyranoside

IRES: Internal ribosome entry site

K_(D): Dissociation constant

k_(off): Dissociation rate

k_(on): Association rate

MHC: Major histocompatibility complex

OPD: O-phenylenediamine

PEG: Polyethylene glycol

scFv: Single-chain variable fragment

SPR: Surface plasmon resonance

TB: Terrific Broth

TCE: T cell epitope

TCR: T cell receptor

TIL: Tumor infiltrating lymphocyte

V_(H): Variable heavy chain

V_(L): Variable light chain

WT1: Wilms tumor protein 1

In the description that follows, certain conventions will be followed asregards the usage of terminology. Generally, terms used herein areintended to be interpreted consistently with the meaning of those termsas they are known to those of skill in the art.

An “antigen-binding protein” is a protein or polypeptide that comprisesan antigen-binding region or antigen-binding portion, that is, has astrong affinity to another molecule to which it binds. Antigen-bindingproteins encompass antibodies, antigen receptors and fusion proteins.

“Antibody” and “antibodies” as those terms are known in the art refer toantigen binding proteins that arise in the context of the immune system.The term “antibody” as referred to herein includes whole, full lengthantibodies and any fragment thereof in which the “antigen-bindingportion” or “antigen-binding region” is retained or single chainsthereof. A naturally occurring “antibody” is a glycoprotein comprisingat least two heavy (H) chains and two light (L) chains inter-connectedby disulfide bonds. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as V_(H)) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as V_(L)) and a light chain constantregion. The light chain constant region is comprised of one domain,C_(L). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is, composed of three CDRsand four FRs arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system.

The term “antigen-binding portion” or “antigen-binding region” of anantibody (or simply “antigen portion”), as used herein, refers to thatregion or portion of the antibody that confers antigen specificity;fragments of antigen-binding proteins, for example, antibodiestherefore, includes one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., an HLA-peptidecomplex). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of antigen-binding fragments encompassed within the term“antibody fragments” of an antibody include a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and CH1 domains; a F(ab)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; a Fd fragment consisting of theV_(H) and CH1 domains; a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody; a dAb fragment (Ward et al.,1989 Nature 341:544-546), which consists of a V_(H) domain; and anisolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An “isolated antibody” or “isolated antigen-binding protein” is onewhich has been identified and separated and/or recovered from acomponent of its natural environment.

Traditionally, the MHC-peptide complex could only be recognized by aT-cell receptor (TCR), limiting our ability to detect an epitope ofinterest to use of T cell-based readout assays. In the presentdisclosure, antigen binding proteins, including antibodies and chimericantigen receptors, having an antigen-binding region based on scFvs thatare selected from human scFv phage display libraries using recombinantHLA-peptide complexes are described. These molecules demonstratedexquisite specificity, for example as shown with anti-EBNA and anti-WT1antigen-binding proteins that recognize only the HLA-A2-LLDFVRFMGV andHLA-A2-RMFPNAPYL complexes, respectively. In addition, along with theirinability to bind to HLA-complexes containing other peptides, themolecules were also unable to bind to the peptides themselves, furtherdemonstrating their TCR-like specificity.

The scFvs of the disclosure selected by phage display were initiallytested for their ability to bind to peptide presented on the surface ofHLA-positive cells. After T2 cells and BLCLs were incubated in thepresence of peptide, the cells could selectively recognize them usingflow cytometry. In the case of one peptide, LLDFVRFMGV (SEQ ID NO:), thecomplex which the peptide formed with HLA could be detected on thesurface of a BLCL even 24 hours after pulsing, further demonstrating theutility of these antibodies.

In some embodiments, the antigen binding proteins of the inventioninclude antibodies that have the scFv sequence fused to the 2^(nd) and3^(rd) constant domains of the heavy chain (CH_(2, 3)), forming thebottom third of the Fc region of a human immunoglobulin to yield abivalent protein and fragments thereof, increasing the overall avidityand stability of the antibody. In addition, the Fc portion allows thedirect conjugation of other molecules, including but not limited tofluorescent dyes, cytotoxins, radioisotopes etc. to the antibody forexample, for use in antigen quantitation studies, to immobilize theantibody for affinity measurements using surface plasmon resonance(SPR), for targeted delivery of a therapeutic agent, to test forFc-mediated cytotoxicity using CD16-expressing immune effector cells andmany other applications.

The purified scFv-Fc fusion proteins were tested for binding to theirtargeted T-cell epitopes (TCEs) by way of ELISA and peptide-pulsed APCs.Once they were validated to maintain their specificity, one molecule,EBNA Clone 315 was used for affinity determination. That this moleculewas able to bind bound to its targeted TCE through a 1:1 interactionwith 10-100 fold greater affinity compared to a typical TCR-MHC-peptidecomplex interaction was demonstrated.

Correlation of peptide pulsing of APCs with antigen density wasdemonstrated. Fluorescently-conjugated scFv-Fc, combined withquantitation beads, allowed the approximation of the number of complexesthat are formed when cells are incubated with different concentrationsof peptide. Using this information, it was possible to approximate thesensitivity of an scFv and scFv-Fc fusion protein to be around 100complexes, using flow cytometry.

Lastly, whether the Fc portion of the fusion protein maintained itseffector function was tested. Using a scFv embodiment of the invention,CD16(V)-transduced NK92MI cells, and peptide-pulsed target cells, it wasdemonstrated that the antibody maintained its Fc-mediated effectorfunctions by way of ADCC.

The results presented here highlight the specificity, sensitivity andutility of the antigen binding proteins of the invention in targetingMHC-peptide complexes.

In one embodiment, therefore, the present invention relates torecombinant antigen-binding molecules and portions thereof thatrecognize a complex of a peptide/protein fragment derived from anintracellular or viral protein, and an MHC class I molecule, forexample, as the complex might be appear at the cell surface forrecognition by a T-cell.

The molecules of the invention are based on the identification andselection of a single chain variable fragment (scFv) using phagedisplay, the amino acid sequence of which confers the molecules'specificity for the MHC restricted peptide of interest and forms thebasis of all antigen binding proteins of the disclosure. The scFv,therefore, can be used to design a diverse array of “antibody”molecules, including, for example, full length antibodies, fragmentsthereof, such as Fab and F(ab′)₂, minibodies, fusion proteins, includingscFv-Fc fusions, multivalent antibodies, that is, antibodies that havemore than one specificity for the same antigen or different antigens,for example, bispecific T-cell engaging antibodies (BiTe), tribodies,etc. (see Cuesta et al., Multivalent antibodies: when design surpassesevolution. Trends in Biotechnology 28:355-362 2010).

In an embodiment in which the antigen-binding protein is a full lengthantibody, the heavy and light chains of an antibody of the invention maybe full-length (e.g., an antibody can include at least one, andpreferably two, complete heavy chains, and at least one, and preferablytwo, complete light chains) or may include an antigen-binding portion (aFab, F(ab′)₂, Fv or a single chain Fv fragment (“scFv”)). In otherembodiments, the antibody heavy chain constant region is chosen from,e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In someembodiments, the immunoglobulin isotype is selected from IgG1, IgG2,IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). The choiceof antibody type will depend on the immune effector function that theantibody is designed to elicit.

In constructing a recombinant immunoglobulin, appropriate amino acidsequences for constant regions of various immunoglobulin isotypes andmethods for the production of a wide array of antibodies are well knownto those of skill in the art.

In some embodiments, the constant region of the antibody is altered,e.g., mutated, to modify the properties of the antibody (e.g., toincrease or decrease one or more of: Fc receptor binding, antibodycarbohydrate, for example glycosylation or fucosylation, the number ofcysteine residues, effector cell function, or complement function).

In one embodiment, the antigen binding protein is an anti-WT1/HLA-A2antibody or fragment thereof having an antigen binding region thatcomprises the amino acid sequence of SEQ ID NO: 2 and specifically bindsto a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) inconjunction with HLA-A2. In other embodiments, the anti-WT-1 antibody isa scFv-Fc fusion protein or full length human IgG with VH and VL regionsor CDRs selected from Table 1.

TABLE 1 Antigen WT1 Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs: 1 2 3 VHSYAMS QIDPWGQETLYADSVKG LTGRFDY (SEQ ID NO. 38) (SEQ ID NO. 40)(SEQ ID NO. 48 ) VL RASQSISSYLN SASQLQS QQGPGTPNT (SEQ ID NO: 56)(SEQ ID NO: 57) (SEQ ID NO. 64) Full VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSQIDPWGQETLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLTGRFDYWGQGTLVTVS (SEQ ID NO: 22) Full VLSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASQLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGPGTPNTFGQGTKVEIKRA (SEQ ID NO: 23) scFvEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSQIDPWGQETLYADSVKGRFTIcloneSRDNSKNTLYLQMNSLRAEDTAVYYCAKLTGRFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQ 45SPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASQLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGPGTPNTFGQGTKVEIKRA (SEQ ID NO: 2) DNAGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG(5′-3′)CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACAGATTGATCCTTGGGGTCAGGAGACATTGTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACTTACTGGTCGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCGGCATCCCAGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGGTCCGGGGACTCCTAATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCC (SEQ ID NO: 3)

In another embodiment, the antigen binding protein is an anti-EBNA3Cantibody or fragment thereof that has an antigen binding region thatcomprises the amino acid sequence of SEQ ID NO: 5 and specifically bindsto a peptide with the amino acid sequence LLDFVRFMGV (SEQ ID NO: 4) inconjunction with HLA-A2. In other embodiments, the anti-EBNA3C antibodyis a scFv-Fc fusion protein or full length human IgG with VH and VLregions or CDRs selected from Table 2.

TABLE 2 Antigen EBNA3C Peptide LLDFVRFMGV (SEQ ID NO: 4 CDRs 1 2 3 VHGYAMS EIAPPGLNTRYADSVKG SDTAFDY (SEQ ID NO: 39) (SEQ ID NO: 41)(SEQ ID NO: 49) VL RASQSISSYLN LASNLQS QQAEYMPLT (SEQ ID NO: 56)(SEQ ID NO: 58) (SEQ ID NO: 65) Full VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSEIAPPGLNTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSDTAFDYWGQGTLVTVS (SEQ ID NO: 24) Full VLSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYLASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAEYMPLTFGQGTKVEIKRA (SEQ ID NO: 25) scFvEVQLLESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSEIAPPGLNTRYADSVKGRFTIScloneRDNSKNTLYLQMNSLRAEDTAVYYCAKSDTAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQS 315PSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYLASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAEYMPLTFGQGTKVEIKRA (SEQ ID NO: 5) DNAGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG(5′-3′)CAGCCTCTGGATTCACCTTTAGCGGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGAGATTGCGCCGCCTGGTTTGAATACACGTTACGCAGACTCCGTGAAGGGCCGGTTCACTATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCGGATACTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCTGGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCGGAGTATATGCCTCTGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCC (SEQ ID NO: 6)

In yet another embodiment, the antigen binding protein is an anti-CCND1antibody or fragment thereof that comprises the amino acid sequence ofone of SEQ ID NOs: 8 or 10 and specifically binds to a peptide with theamino acids sequence RLTRFLSRV (SEQ ID NO: 7) in conjunction withHLA-A2. In other embodiments, the anti-CCND1 antibody is a scFv-Fcfusion or full length human IgG with VH and VL regions or CDRs selectedfrom Tables 3 and 4.

TABLE 3 Antigen CCND1 Peptide RLTRFLSRV (SEQ ID NO. 7) CDRs 1 2 3 VHSYAMS(38) TISDSDATDYADSVKG(42) TTDYFDY(50) VL RASQSISSYLN(56)YASYLQS(59) QQSSSSPDT(66) Full VHEVQLLESGGGLVQPGGSLRLSCATSGFTFSSYAMSWVRQAPGKGLEWVSTISDSDATDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTIDYFDYWGQGTLVIVS(26) Full VLSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSSPDTFGQGTKVEIKRAA(27) scFvEVQLLESGGGLVQPGGSLRLSCATSGFTFSSYAMSWVRQAPGKGLEWVSTISDSDATDYADSVKGRFTISRclone 5,DNSKNTLYLQMNSLRAEDTAVYYCAKTTDYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSP 17SSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSSPDTFGQGTKVEIKRAA(8) DNAGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG(5′-3′)CAACCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTTCTGATAGTGATGCTACAGATTACGCAGACTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTACTGATTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTATGCATCCTATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTTCTAGTTCTCCTGATACGTICGGCCAAGGGACCAAGGIGGAAATCAAACGGGCGGCC(9)

TABLE 4 Antigen CCND1 Peptide RLTRFLSRV(SEQ ID NO: 7) CDRs: 1 2 3 VHSYAMS(38) DISDDGDATYYADSVKG(43) SSTTFDY(51) VL RASQSISSYLN(56)AASALQS(60) QQGTDSPAT(67) Full VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDISDDGDATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSTTFDYWGQGTLVTVS(28) Full VLSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGTDSPATFGQGTKVEIKRAA(29) scFvEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDISDDGDATYYADSVKGRFTIcloneSRDNSKNTLYLQMNSLRAEDTAVYYCAKSSTTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQ 43SPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGTDSPATFGQGTKVEIKRAA(10) DNAGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG(5′-3′)CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGATATTTCTGATGATGGTGATGCTACATATTACGCAGACTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTTCTACTACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCGCCTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGGTACTGATAGTCCTGCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCC(11)

In yet another embodiment, the antigen binding protein is an anti-HUDantibody or fragment thereof that comprises the amino acid sequence ofone of SEQ ID NOs: 13, 14 and 17 and has an antigen-binding region thatspecifically binds to a peptide with the amino acid sequence RIITSTILV(SEQ ID NO: 12) in conjunction with HLA-A2. In other embodiments, theanti-HUD antibody is a scFv-Fc fusion protein or full length human IgGwith VH and VL regions or CDRs selected from Tables 5-7.

TABLE 5 Antigen HUD Peptide RIITSTILV(SEQ ID NO: 12) CDRs: 1 2 3 VHSYAMS(38) DIASTGYYTDYADSVKG(44) NNASFDY(52) VL RASQSISSYLN(56)DASTLQS(61) QQTDSYPTT(68) Full VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDIASTGYYTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNNASFDYWGQGTLVTVS(30) Full VLSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTDSYPTTFGQGTKVEIKR(31) scFvEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDIASTGYYTDYADSVKGRFTIScloneRDNSKNTLYLQMNSLRAEDTAVYYCAKNNASFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQ H128SPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTDSYPTTFGQGTKVEIKR(13) DNAGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG(5′-3′)CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCGGATATTGCTTCTACTGGTTATTATACAGATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATAATGCTAGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACTGATTCTTATCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG(15)

TABLE 6 Antigen HUD Peptide RIITSTILV(SEQ ID NO: 12) CDRs: 1 2 3 VHSYAMS(38) SISSSGYYTDYADSVKG(45) SASSFDY(53) VL RASQSISSYLN(56)DASTLQS(61) QQDDAYPTT(69) Full VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSSGSYTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSASSFDYWGQGTLVTVS(32) Full VLSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDDAYPTTFGQGTKVEIKR(33) scFvEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSSGSYTDYADSVKGRFTIScloneRDNSKNTLYLQMNSLRAEDTAVYYCAKSASSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQS H78PSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDDAYPTTFGQGTKVEIKR(14) DNAGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG(5′-3′)CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTAGTTCTGGTAGTTATACAGATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTGCTTCTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGATGATGCTTATCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG(16)

TABLE 7 Antigen HUD Peptide RIITSTILV(SEQ ID NO: 12) CDRs: 1 2 3 VHSYAMS(38) SISSDGSYTDYADSVKG(46) STDAFDY(54) VL RASQSISSYLN(56)AASYLQS(62) QQDNNYPTT(70) Full VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSDGSYTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTDAFDYWGQGTLVTVS(34) Full VLSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQDNNYPTTFGQGTKVEIKR(35) scFvEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSDGSYTDYADSVKGRFTIScloneRDNSKNTLYLQMNSLRAEDTAVYYCAKSTDAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSH110PSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQDNNYPTTFGQGTKVEIKR(17) DNAGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG(5′-3′)CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTTCTTCTGATGGTAGTTATACAGATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTACTGATGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCTATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCTCTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGATAATAATTATCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG(18)

In yet another embodiment, the antigen binding protein is an anti-cdr2antibody or fragment thereof that comprises the amino acid sequence ofSEQ ID NO: 20 and specifically binds to a peptide with amino acidsLLEEMFLTV (SEQ ID NO: 19) in conjunction with HLA-A2. In otherembodiments, the anti-cdr2 antibody is a scFv-Fc fusion protein or fulllength human IgG with VH and VL regions or CDRs selected from Table 8.

TABLE 8 Antigen cdr2 Peptide LLEEMFLTV(SEQ ID NO: 19) CDRs: 1 2 3 VHSYAMS(38) TINYSGSGTTYADSVKG(47) NAAYFDY(55) VL RASQSISSYLN(56)GASGLQS(63) QQSANAPTT(71) Full VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINYSGSGTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNAAYFDYWGQGTLVTVS(36) Full VLSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSANAPTTFGQGTKVEIKR(37) scFvEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINYSGSGTTYADSVKGRFTIScloneRDNSKNTLYLQMNSLRAEDTAVYYCAKNAAYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQS L9PSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSANAPTTFGQGTKVEIKR(20) DNAGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG(5′-3′)CAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAATTATTCTGGTTCTGGTACAACTTACGCAGACTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATGCTGCTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCGGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTGCTAATGCTCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGG(21)

Embodiments of the antigen-binding proteins of the disclosure inaccordance with Tables 1-8 include, but are not limited to thefollowing:

an anti-WT-1 antibody which binds to an HLA-restricted peptide RMFPNAPYL(SEQ ID NO: 1) comprising: (i) an HVR-L1 sequence of RASQSISSYLN (SEQ IDNO: 56) (ii) an HVR-L2 sequence of SASQLQS (SEQ ID NO: 57) (iii) anHVR-L3 sequence of QQGPGTPNT (SEQ ID NO: 64) (iv) an HVR-H1 sequence ofSYAMS (SEQ ID NO: 38) (v) an HVR-H2 sequence of QIDPWGQETLYADSVKG (SEQID NO: 40), and (vi) an HVR-H3 sequence of LTGRFDY (SEQ ID NO: 48);

an anti-EBNA3C antibody which binds to HLA-A2 restricted peptideLLDFVRFMGV (SEQ ID NO: 4) comprising: (i) an HVR-L1 sequence ofRASQSISSYLN (SEQ ID NO: 56) (ii) an HVR-L2 sequence of LASNLQS (SEQ IDNO: 58) (iii) an HVR-L3 sequence of QQAEYMPLT (SEQ ID NO: 65) (iv) anHVR-H1 sequence of GYAMS (SEQ ID NO: 39) (v) an HVR-H2 sequence ofEIAPPGLNTRYADSVKG (SEQ ID NO: 41), and (vi) an HVR-H3 sequence ofSDTAFDY (SEQ ID NO: 49);

an anti-CCND1 antibody which binds to HLA-A2 restricted peptideRLTRFLSRV (SEQ ID NO: 7) comprising: (i) an HVR-L1 sequence ofRASQSISSYLN (SEQ ID NO: 56) (ii) an HVR-L2 sequence of YASYLQS (SEQ IDNO: 59) (iii) an HVR-L3 sequence of QQSSSSPDT (SEQ ID NO: 66) (iv) anHVR-H1 sequence of SYAMS (SEQ ID NO: 38) (v) an HVR-H2 sequence ofTISDSDATDYADSVKG (SEQ ID NO: 42), and (vi) an HVR-H3 sequence of TTDYFDY(SEQ ID NO: 50);

an anti-CCND1 antibody which binds to HLA-A2 restricted peptideRLTRFLSRV (SEQ ID NO: 7) comprising: (i) an HVR-L1 sequence ofRASQSISSYLN (SEQ ID NO: 56) (ii) an HVR-L2 sequence of AASALQS (SEQ IDNO: 60) (iii) an HVR-L3 sequence of QQGTDSPAT (SEQ ID NO: 67) (iv) anHVR-H1 sequence of SYAMS (SEQ ID NO: 38) (v) an HVR-H2 sequence ofDISDDGDATYYADSVKG (SEQ ID NO: 43), and (vi) an HVR-H3 sequence ofSSTTFDY (SEQ ID NO: 51);

an anti-HUD antibody which binds to HLA-A2 restricted peptide RIITSTILV(SEQ ID NO: 12) comprising: (i) an HVR-L1 sequence of RASQSISSYLN (SEQID NO: 56) (ii) an HVR-L2 sequence of DASTLQS (SEQ ID NO: 61) (iii) anHVR-L3 sequence of QQTDSYPTT (SEQ ID NO: 68) (iv) an HVR-H1 sequence ofSYAMS (SEQ ID NO: 38) (v) an HVR-H2 sequence of DIASTGYYTDYADSVKG (SEQID NO: 44), and (vi) an HVR-H3 sequence of NNASFDY (SEQ ID NO: 52);

an anti-HUD antibody which binds to HLA-A2 restricted peptide RIITSTILV(SEQ ID NO: 12) comprising: (i) an HVR-L1 sequence of RASQSISSYLN (SEQID NO: 56) (ii) an HVR-L2 sequence of DASTLQS (SEQ ID NO: 61) (iii) anHVR-L3 sequence of QQDDAYPTT (SEQ ID NO: 69) (iv) an HVR-H1 sequence ofSYAMS (SEQ ID NO: 38) (v) an HVR-H2 sequence of SISSSGYYTDYADSVKG (SEQID NO: 45), and (vi) an HVR-H3 sequence of SASSFDY (SEQ ID NO: 53);

an anti-HUD antibody which binds to HLA-A2 restricted peptide RIITSTILV(SEQ ID NO: 12) comprising: (i) an HVR-L1 sequence of RASQSISSYLN (SEQID NO: 56) (ii) an HVR-L2 sequence of AASYLQS (SEQ ID NO: 62) (iii) anHVR-L3 sequence of QQDNNYPTT (SEQ ID NO: 70) (iv) an HVR-H1 sequence ofSYAMS (SEQ ID NO: 38) (v) an HVR-H2 sequence of SISSDGSYTDYADSVKG (SEQID NO: 46), and (vi) an HVR-H3 sequence of STDAFDY (SEQ ID NO: 54); and

an anti-cdr2 antibody which binds to HLA-A2 restricted peptide LLEEMFLTV(SEQ ID NO: 19) comprising: (i) an HVR-L1 sequence of RASQSISSYLN (SEQID NO: 56) (ii) an HVR-L2 sequence of GASGLQS (SEQ ID NO: 63) (iii) anHVR-L3 sequence of QQSANAPTT (SEQ ID NO: 71) (iv) an HVR-H1 sequence ofSYAMS (SEQ ID NO: 38) (v) an HVR-H2 sequence of TINYSGSGTTYADSVKG (SEQID NO: 47), and (vi) an HVR-H3 sequence of NAAYFDY (SEQ ID NO: 55).

EXAMPLES General Procedures Example 1 Production of BiotinylatedMHC-Peptide Complexes

Soluble MHC class I/peptide complexes were generated by overexpressionof the HLA-A2 heavy chain (HC) and β2 microglobulin (β₂M) as recombinantproteins in E. coli and subsequent in vitro refolding and assembly inthe presence of high concentrations of specific peptide (35, 36). Toobtain soluble MHC/peptide complexes the HC sequence was mutagenized toremove the cytosolic and transmembrane regions. In order to specificallybiotinylate refolded, monomeric MHC/peptide complexes, the HC wasexpressed as a fusion protein containing a specific biotinylation siteat the C-terminus (37, 38). These short sequences are sufficient forenzymatic in vitro biotinylation of a single lysine residue within thissequence using the biotin protein ligase BirA (39). This procedure wascarried out by the MSKCC Tetramer Core Facility.

Example 2 Selection of Phage on Biotinylated MHC-Peptide Complexes

Ex. 2.1: Selection of Phage on HLA-A2/EBNA3C (EBNA) Complex

The Tomlinson I+J human scFv phage display libraries (40), containingapproximately 2.85×10⁸ independent scFv clones, were used for selectionaccording to previously published methods (22) with modifications.7.5×10¹² Phage, from the combination of both libraries, were firstpreincubated with streptavidin paramagnetic Dynabeads (30 μl; Dynal,Oslo, Norway) and 150 μg unbiotinylated HLA-A2-YVDPVITSI (SEQ ID NO:)(irrelevant complex) in 1 ml of PBS to remove any phage which expressedan antibody that binds to streptavidin or the general framework ofHLA-A2.

The dynabeads were subsequently captured using a magnet and thesupernatant (phage and irrelevant complex mixture) transferred to aseparate tube containing 7.5 μg of biotinylated HLA-A2-LLDFVRFMGV(Epstein-Barr virus EBNA3C-derived) and 7.5 μg of biotinylatedHLA-A2-NLVPMVATV (Cytomedullovirus pp65-derived) and incubated at RT for1 hour. The final mixture (1 ml) was then added to 200 μl of Dynabeads(preincubated with 2% Milk and washed with PBS) and the contents weremixed for 15 min. at RT with continuous rotation. The beads were thenwashed 10 times with PBS/0.1% Tween and 3 times with PBS and the boundphage were eluted from the Dynabeads using 1 mg/ml trypsin in PBS (0.5ml) for 15 min. at RT.

The phage were then used to infect TG1 E. coli (growing in log phase) at37° C. in 20 ml of LB for 1 hour. 10¹² KM13 helper phage wassubsequently added to the mixture, further incubated for an additional30 minutes, and the cells pelleted using centrifugation (3000 rpm for 10min.). The resulting cell pellet was resuspended in 200 ml LB+Ampicillin(100 μg/ml)+Kanamycin (50 μg/ml) and incubated overnight at 30° C.

The following morning, the overnight cultures were centrifuged at 3000rpm for 15 min. and the supernatant (180 ml) was mixed with polyethyleneglycol (PEG) on ice for 1 hour so as to precipitate the amplified phagefrom the previous round of selection. The PEG/phage mixture was thencentrifuged at 3000 rpm for 20 min., and some of the resulting phagepellet used for subsequent rounds of panning while the rest was frozendown in 15% glycerol at ˜80° C. Subsequent rounds of panning were doneusing the same protocol as above with an increase in Dynabead washingsteps and a decrease in the amount of biotinylated complexes used forselection.

After the final round of antibody selection (3^(rd) or 4th round), theeluted phage were used to infect both TG1 and HB2151 E. coli; TG1 cellswere cultured overnight as mentioned above while the HB2151 cells wereplated on TYE+Ampicillin (100 μg/ml) agar plates. The next morning,individual colonies from the agar plate were picked and used toinoculate individual wells of a 48-well plate containing 400 μlLB+Ampicillin (100 μg/ml)/well. After incubation for 3-6 hours at 37°C., 200 μl of 50% glycerol solution was added to each well and theplates stored at −80° C. as monoclonal stock cultures.

Ex. 2.2: Selection of Phage on HLA-A2-RMFPNAPYL (WT-1) Complex

Selection was done similarly to the method above with slightmodifications. 3.7×10¹² Phage from the combination of both libraries,were first preincubated with streptavidin paramagnetic Dynabeads (50 μl;Dynal, Oslo, Norway) and 20 μg unbiotinylated HLA-A2-NLVPMVATV(irrelevant complex) in 1 ml of PBS to deplete the streptavidin andHLA-A2 binders. The dynabeads were subsequently captured using a magnetand the supernatant (phage and irrelevant complex mixture) transferredto a separate tube containing 5 μg of biotinylated HLA-A2-RMFPNAPYL(WT1-derived) and incubated at RT for 1 hour. The final mixture (1 ml)was then added to 100 μl of Dynabeads (preincubated with 2% Milk andwashed with PBS) and the contents were mixed for 30 min. at RT withcontinuous rotation. The beads were then washed 10 times with PBS/0.1%Tween and 3 times with PBS and the bound phage were eluted from theDynabeads using 1 mg/ml trypsin in PBS (0.5 ml) for 20 min. at RT. Allsubsequent steps were performed as above.

Example 3 Expression and Purification of Soluble scFv from HB2151

Using the monoclonal glycerol stocks containing individual HB2151clones, separate 48-well plates containing 400 μl LB+Ampicillin (100μg/ml)/well were inoculated in a replica-plate type format using sterilepipette tips. The 48-well culture plates were subsequently incubated at37° C. until the majority of the wells reached an OD600 of 0.4. 200 μlLB+Ampicillin (100 μg/ml)+isopropyl-1-thio-β-D-galactopyranoside (IPTG;1 mM final concentration) was subsequently added to each well to inducescFv production and the plates were further incubated overnight at 28°C. The next morning, the plates were centrifuged at 3000 rpm for 15 min.and the supernatant used for scFv screening.

For large scale expression and purification, monoclonal glycerol stockswere used to inoculate 3 ml of Terrific Broth (TB) and incubated at 37°C. until an OD600 of 0.8 was reached. Each 3 ml culture was subsequentlydivided amongst four flasks, each containing 250 ml TB+Ampicillin (100μg/ml). The cultures were then incubated at 37° C. until an OD600 of0.4-0.5 was reach, after which IPTG was added to a final concentrationof 0.5 mM and the cultures incubated overnight at 30° C. The nextmorning, the overnight cultures were centrifuged at 4000 rpm for 25 min.The supernatant was discarded and the pellets dissolved in 50 ml PBS+10mM imidazole. The cell suspensions were passed through a cellhomogenizer (5000 pounds per square inch) and the resulting cell lysateswere centrifuged at 12,000 rpm for 15 min. The supernatants were thenpassed over a 0.22 μm filter pre-layered with diatomaceous earth and theresulting filtrates loaded over Vivapure maxiprepMC Nickel affinitycolumns (Sartorius Stedim Biotech, Aubagne, France) using centrifugation(100 rpm for 5 min.). The columns were then washed 4 times using 10 mlPBS+30 mM imidazole (500 rpm for 3 min.) and the scFvs eluted using 20ml PBS+300 mM imidazole (500 rpm for 3 min.). The eluted scFvs wereconcentrated using 10,000 molecular weight cut-off membrane Vivaspincentrifuge tubes at 3000 rpm for 30 min. (Sartorius Stedim Biotech) anddialyzed back into regular PBS. The final scFv products weresubsequently stored at −80° C.

Example 4 Construction of scFv-Fc Fusion Protein and Expression in DG44CHO Cells

Using a proprietary antibody expression vector (referred to herein asIgG Vector), similar to that of pFUSE—hlgG1-Fcl (InvivoGen; San Diego,Calif.), the construct was first modified to contain the CH₂, and CH₃domains of a human IgG₁ (scFv-Fc Vector). Subsequently, the EBNA Clone315 and WT1 Clone 45 scFv sequences were PCR amplified to contain therequired NheI and ApaI restriction sites which would be compatible withthe scFv-Fc vector. The resulting scFv PCR products and antibodyexpression plasmid were digested using the above enzymes (NheI at 37° C.for 2 hours and ApaI at 25° C. for 2 hours) and then ligated together.The ligation products were then transformed into E. coli, plated onTYE+Amplicilin (100 μg/ml), colonies were picked and their plasmidssequenced at the MSKCC Sequencing Core Facility. Once the sequences werevalidated to have the correct scFv sequences upstream of the human IgG₁CH₂ and CH₃ domains, the DNA (5-6 μg was electroporated (AmaxaNucleofactor; Lonza, Switzerland) into 5×10⁶ DG44 Chinese Hamster Ovary(CHO) Cells (Invitrogen) using Program U-030 and 100 μl Solution V. Thecells were then cultured in OptiCHO media (Invitrogen) containing G418(500 μg/ml; added 7 days post-electroporation) at a cell density of1-5×10⁶ DG44 per ml of media. The cells were then expanded toapproximately 700 ml of culture media, which was centrifuged to removethe cells and supernatant used for antibody purification.

Example 5 Expression and Purification of Soluble scFv-Fc Fusion Protein

DG44 supernatant containing the soluble scFv-Fc fusion protein waspurified using the KappaSelect affinity chromatograph medium (GEHealthcare). First, 1.5 ml of KappaSelect resin was loaded onto a columnand activated with 20 ml of PBS. The supernatant was loaded onto thecolumn using a peristaltic pump at a flow rate of approximately 1ml/min. The column was subsequently washed using 40 ml of PBS until theflow-thru registered an OD280 of less than 0.05. The scFv-Fc fusionprotein was then eluted from the resin using 10 ml citrate buffer (pH2.0) and directly into 10 ml of 1 M Tris for neutralization. The elutedscFv-Fc was subsequently concentrated using a 50,000 MWCO Vivaspincentrifuge tube (Sartorius Stedim) and tested for its ability to bind torecombinant antigen using ELISA and the Biacore T100 (GE Healthcare) aswell as natively presented peptide on the surface of T2 cells using flowcytometry.

Example 6 Monoclonal ELISA with Bacterial Phage Clones and Purified scFvand scFv-Fc

Vinyl flat bottom microtiter plates (Thermo Fisher) were used for ELISAassays. Plates were initially coated overnight at 4° C. with BSA-biotin(10 μg/ml; 50 μl/well). The next morning, the contents were discardedand the plates incubated at RT with streptavidin (10 μg/ml; 50 μl/well)for 1 hour. The contents were discarded and the plates incubated withrecombinant biotinylated HLA-A2-peptide complexes (5 μg/ml; 50 μl/well)at RT for 1 hour. The plates were then incubated with 2% Milk (150μl/well) at RT for 1 hour. After blocking, the plates were washed 2times with PBS and then incubated with bacterial supernatant from theirrespective HB2151 culture plate wells, purified scFv, or purifiedscFv-Fc at RT for 1 hour. The contents were discarded, the plates washed5 times with PBS, and then incubated at RT for 1 hour with either amouse-anti-myc tag antibody (Clone 9E10; Sigma Aldrich. 0.5 μg/ml; 100μl/well in 0.5% Milk) to detect the scFv or a goat-anti-human-HRP(Jackson Immunoresearch Laboratories. 0.5 μg/ml; 100 μl/well in 0.5%Milk) to detect the scFv-Fc. The contents were discarded, the plateswashed 5 times with PBS, and those receiving the scFv were furtherincubated with a goat-anti-mouse-HRP (Jackson ImmunoresearchLaboratories. 0.5 μg/ml; 100 μl/well in 0.5% Milk) at RT for 1 hourwhile the plates receiving the scFv-Fc were developed usingo-phenylenediamine (OPD) buffer (150 μl/well), which was made bycombining 20 mg of OPD tablets in 40 ml of citrate phosphate buffer with40 μl 30% hydrogen peroxide. The color reaction was stopped by adding 30μl of 5N sulfuric acid to each well and the plates read using the DynexMRX ELISA plate reader at 490 nm. Lastly, the contents of the scFvplates were discarded, the plates washed 5 times with PBS, and developedaccording to the method above.

Example 7 Cell Lines and Peptides

Tap-deficient HLA-A2⁺ T2 cells, 6268A, GKO (both HLA-A2), DIMT and JG19(both HLA-A2⁺) B-cell lymphoblastic cell lines (BLCLs) were used forantigen presentation studies. Cells were normally cultured in RPMI1640+10% Fetal Bovine Serum (FBS). For antigen presentation, T2 cellswere harvested and transferred to serum-free IMDM+10 μg/mlβ2-microglobulin (β2M). The T2 cells would then be incubated with 20 μMor less of either LLDFVRFMGV-peptide (derived from EBNA3C) or any numberof irrelevant peptides at 37° C. for 5 hours. Studies with BLCLs weredone in the same manner as with T2 cells with the occlusion of β₂M inthe media. Pulse-Chase experiments with DIMT BLCLs were done by firstpulsing the BLCLs in serum-free IMDM with 20 μM LLDFVRFMGV for 5 hoursat 37° C. The cells were then washed with fresh RPMI 1640+10% FBS,transferred back into this culture medium and cultured further at 37° C.for 5 and 24 hours, followed by flow cytometric analysis at each timepoint using EBNA Clone 315 scFv.

Example 8 Binding Kinetics Analysis

Kinetic measurements were performed by surface plasmon resonance usingthe BIAcore T100 (GE Biosciences). Briefly, the first two flow cells ofa CM5 chip (GE Biosciences) were activated using the standard aminecoupling reagents in HBS-EP running buffer (0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.005% Tween 20) with flow cell 2 immobilized with the purifiedEBNA Clone 315 scFv-Fc fusion protein using 10 mM Acetate (pH 5).Subsequently, the target HLA-A2-peptide monomer (222 nM-13.875 nM) wasinjected over both the 1st (reference) and 2nd flow cells at 20 μl/min.for 120 sec., followed by the addition of running buffer for an extra180 sec. Kinetics values were determined using the BIAcore T100Evaluation Software 2.0 and 1:1 binding model (local Rmax).

Example 9 Flow Cytometry

Peptide-pulsed T2 cells and BLCLs were transferred to plasticpolystyrene round-bottom tubes (Becton Dickinson Labware) and washedwith PBS. The cells were subsequently incubated with 5 μg of eithertargeted or non-specific purified scFv or scFv-Fc on ice for 40 min. Thecells were washed with PBS and then incubated with 1 μg of biotinylatedmouse-anti-myc antibody (Clone 9E10; Sigma Aldrich) or biotinylatedmouse-anti-human IgG Fc-specific (Jackson Immunoresearch Laboratories)on ice for 30 min. The cells were washed with PBS and then incubatedwith streptavidin-PE (BD Biosciences). Lastly, the cells were washedonce more with PBS and analyzed on the BD FACS Calibur.

For CD107a cytotoxicity assays, transduced-NK92MI cells and target T2cells were cocultured in a 1:1 effector:target (E:T) ratio (2.5-5.0×10⁵cells each) in 200 μl complete Alpha Essential medium (12.5% horse serumand 12.5% FBS) (Invitrogen)+10-15 μl anti-CD107a-PE at 37° C. for 5Hours. The cell mixture was then washed with PBS and analyzed on the BDFACS Caliber.

For FACS sorting experiments, retrovirally transduced NK92MI cells weresorted based on GFP intensity using the BD Aria Flow Cytometer under theguidance of the MSKCC Flow Cytometry facility.

Example 10 Quantitation of HLA-A2-LLDFVRFMGV Complexes on Peptide-PulsedT2 Cells

For MHC-peptide complex quantitation, the EBNA Clone 315 scFv-Fc wasfirst directly conjugated to Alexa Fluor 647 using the APEX Alexa Fluor647 Antibody Labeling Kit (Invitrogen). The kit yields about 10-20 μg oflabeled antibody.

For quantitation, the Quantum Simply Cellular anti-Human IgG kit wasused (Bangs Laboratories) along with the technical assistance ofHong-fen Guo in our laboratory. Briefly, the kit is comprised of fivemicrosphere populations; one blank and four labeled with increasingamounts of anti-human IgG. The beads and the peptide pulsed T2 cells(37° C. for 5 hours) were then labeled with the same fluorescentlyconjugated EBNA Clone 315 scFv-Fc on ice for 30 minutes. The cells werethen washed with PBS and analyzed on the BD FACS Calibur along with thelabeled beads. The Excel-based QuickCal analysis template that'sprovided with each kit aids in correlating fluorescence intensity withantigen density on the T2 cells. Each of the 4 data points are theaverage of duplicates.

Example 11 Construction of the WT-1 Clone 45 Chimeric Antigen Receptor

The original chimeric antigen receptor was obtained from Dr. DarioCampana from St. Jude Children's Hospital and previously described (41).For future compatibility purposes, a scFv-CD3ζ-4-1BB DNA construct(similar to that seen in the original chimeric immune receptor, withEcoRI and XhoI flanking the 5′ and 3′ ends) was purchased (pUC57 vectorfrom Genescript; Piscataway, N.J.) and contained an irrelevant scFvflanked by SfiI and NotI. The plasmid containing the EBNA Clone 315 scFvsequence (pIT2 vector from the Tomlinson library) was purified (Qiagenminiprep DNA isolation kit) from overnight culture of the bacterialstock in LB+Amplicilin (100 μg/ml). The scFv sequence was excised fromthe pIT2 vector using SfiI (50° C. for 2 hours) and NotI (37° C. for 2hours) inserted into the purchased and predigested (SfiI and NotI) pUC57vector. After ligation, the product was transformed into NEB 5-alphacompetent E. coli (New England Biolabs), the cells plated onTYE+Amplicilin (100 μg/ml), colonies were picked and cultured inLB+Amplicilin (100 μg/ml), their plasmids purified and the product sizeswere verified by gel electrophoresis. Plasmids which were found to havethe correct ligation products were subsequently excised from the pUC57vector using EcoRI (37° C. for 2 hours) and XhoI (37° C. for 2 hours)and used for insertion into the vector provided to us by the Campanalaboratory. The ligation products were then transformed into E. coli asabove, plated on TYE+Amplicilin (100 μg/ml), colonies were picked andtheir plasmids sequenced using the reverse primer 788A(5′-CCCTTGAACCTCCTCGTTCGACC-3′) (SEQ ID NO: 72) at the MSKCC SequencingCore Facility. Once the sequences were validated, the DNA was packagedinto retrovirus and used to infect NK92MI cells.

Example 12 Construction of the EBNA Clone 315 Chimeric Antigen Receptor

Due to compatibility issues, the pUC57 scFv-CD3ζ-4-1BB DNA constructpurchased from Genescript and mentioned above was used to replace theWT1 Clone 45 scFv with the EBNA Clone 315 scFv. First, the plasmidcontaining the EBNA Clone 315 scFv sequence (pIT2 vector from theTomlinson library) was purified (Qiagen miniprep DNA isolation kit) fromovernight culture of the bacterial stock in LB+Amplicilin (100 μg/ml).The scFv sequence was excised from the pIT2 vector using SfiI (50° C.for 2 hours) and NotI (37° C. for 2 hours) and ligated to thepredigested (SfiI and NotI) pUC57 vector. After ligation, the productwas transformed into E. coli, colonies were picked, cultured overnight,their plasmids purified and the product sizes verified by gelelectrophoresis. Plasmids which were found to have the correct ligationproducts were subsequently excised from the pUC57 vector using EcoRI(37° C. for 2 hours) and XhoI (37° C. for 1 minute). Due to the presenceof a XhoI site inside of the EBNA Clone 315 scFv sequence, the DNA waspartially digested with XhoI and then completely digested using EcoRI.This allowed for the isolation of the correct DNA fragment which keptthe integrity of the scFv sequence while removing the entire CARsequence from the pUC57 vector. After insertion into the vector providedto us by the Campana laboratory, the ligation products were thentransformed into E. coli as above, plated on TYE+Amplicilin (100 μg/ml),colonies were picked and their plasmids sequenced using the reverseprimer 788A (5′-CCCTTGAACCTCCTCGTTCGACC-3′) (SEQ ID NO: 72) at the MSKCCSequencing Core Facility. Once the sequences were validated, the DNA waspackaged into retrovirus and used to infect NK92MI cells.

Example 13 Retroviral Production, DNA Packaging, and Infection of NK92MICells

To produce CAR-containing retrovirus, the following procedure wasemployed which used a 293T-based retroviral production cell line (GP2).Briefly, 7 μg of CAR DNA was combined with 3.5 μg of PCLAmpho helperconstruct and 3.5 μg pVSVg in 1 ml of serum-free DMEM. This mixture wasthen combined with 1 ml serum-free DMEM containing 36 μl ofLipofectamine 2000 (Invitrogen) and incubated at RT for 20 min.Afterwards, the DNA-Lipofectamine complex (2 ml) was mixed with GP2cells (3-5×10⁶) in 10 ml of DMEM+10% FBS and cultured at 37° C. for 72hours. Subsequently, the supernatant (12 ml) was depleted of GP2 cellsduring recovery and incubated with 3 ml Lenti-X Concentrator solution(Clontech) at 4° C. for 12-16 hours. Afterwards, the solution wascentrifuged at 3000 rpm for 15 min., the supernatant discarded, and thepellet dissolved in 1 ml complete Alpha Essential medium containing5×10⁵ NK92MI cells. The cells were then incubated for 72 hours andchecked by flow cytometry for CAR expression via GFP (the CAR gene isexpressed under a CMV promoter which is followed by IRES-GFP).

Example 14 Construction of scFv-Fc Fusion Protein and Expression in DG44CHO Cells

Using a proprietary antibody expression vector similar to that ofpFUSE—hlgG1-Fc1 (Invivogen; San Diego, Calif.), the Clone 315 scFvsequence was first PCR amplified to contain the required NheI and ApaIrestriction sites. The resulting PCR product and expression plasmid weredigested using the above enzymes (NheI at 37° C. for 2 hours and ApaI at25° C. for 2 hours) and ligated together. The ligation products werethen transformed into E. coli, plated on TYE+Amplicilin (100 μg/ml),colonies were picked and their plasmids sequenced at the MSKCCSequencing Core Facility. Once the sequences were validated to have theClone 315 scFv sequence upstream of the human IgG₁ CH2 and CH3 domains,the DNA was electroporated (Amaxa Nucleofactor; Lonza, Switzerland) into5×10⁶ DG44 Chinese Hamster Ovary (CHO) Cells (Invitrogen) using ProgramU-030 and 100 μl Solution V. The cells were then cultured in OptiCHOmedia (Invitrogen) containing G418 (500 μg/ml; added 7 dayspost-electroporation) at a cell density of 1-5×10⁶ DG44 per ml of media.

Example 15 Retro Viral Production, DNA Packaging, and Infection ofNK92MI Cells

To produce CAR-containing retrovirus, the following procedure wasemployed which used a 293T-based retroviral production cell line (GP2).Briefly, 7 μg of CAR DNA was combined with 3.5 μg of PCLAmpho helperconstruct and 3.5 μg pVSVg in 1 ml of serum-free DMEM. This mixture wasthen combined with 1 ml serum-free DMEM containing 36 μl ofLipofectamine 2000 (Invitrogen) and incubated at RT for 20 min.Afterwards, the DNA-Lipofectamine complex (2 ml) was mixed with GP2cells (3-5×10⁶) in 10 ml of DMEM+10% FBS and cultured at 37° C. for 72hours. Subsequently, the supernatant (12 ml) was depleted of GP2 cellsduring recovery and incubated with 3 ml Lenti-X Concentrator solution(Clontech) at 4° C. for 12-16 hours. Afterwards, the solution wascentrifuged at 3000 rpm for 15 min., the supernatant discarded, and thepellet dissolved in 1 ml complete Alpha Essential medium containing5×10⁵ NK92MI cells. The cells were then incubated for 72 hours andchecked by flow cytometry for CAR expression via GFP (the CAR gene isexpressed under a CMV promoter which is followed by IRES-GFP).

Example 16 ⁵¹Cr Release Cytotoxicity Assay

The capacity of CAR equipped NK92MI cells to lyse BLCLs was evaluatedusing a ⁵¹Chromium release assay. Briefly, peptide pulsed or unpulsed⁵¹Cr-labeled BLCLs were plated in round-bottom 96-well plates (5×10³cells/well) in RPMI 1640 with 10% FBS. Subsequently, CAR equipped NK92MIcells were added to the BLCL containing wells at different effector(E)/target (T) ratios and incubated for 4 hours at 37° C., after whichthe cultures were depleted of cells and ⁵¹Cr-release was measured in thesupernatants. All E:T ratios were done in triplicate, with the averageplotted on the graphs. % ⁵¹Cr Release was determined using the followingformula: ((Sample Release−Spontaneous Release)/(TotalRelease−Spontaneous Release))×100.

Example 17 Affinity Selection of Phage on Virally-Derived RecombinantHLA-A2-Peptide Complexes

Biotinylated and non-biotinylated recombinant HLA-A2-peptide complexespresenting various different peptides previously shown to bind to HLA-A2were obtained from the MSKCC Tetramer Core Facility. For selectionpurposes, the Tomlinson I and J phage display libraries were combinedand first preincubated with non-biotinylated, irrelevantHLA-A2-YVDPVITSI complex along with streptavidin paramagnetic beads sothat any phage which expresses an antibody that may bind to the generalframework of HLA-A2, or the streptavidin beads themselves, areeventually discarded during the washing steps. Subsequently, thecontents (phage and irrelevant complex) were incubated with biotinylatedHLA-A2-LLDFVRFMGV (EBNA3C) and biotinylated HLA-A2-NLVPMVATV (pp65)simultaneously in equimolar ratios and captured using streptavidinparamagnetic beads. Once the beads were bound to the biotinylatedcomplexes, the beads were washed with PBS containing Tween 20 and thebound phage were eluted from the beads using trypsin. After twoadditional rounds of selection, the recovered phage were used to infectHB2151 E. coli and plated on ampicillin-containing agar. The nextmorning, individual colonies were picked, cultured overnight in 48-wellculture plates, and their supernatants tested for the presence of scFvon 96-well ELISA plates pre-coated with recombinant HLA-A2-peptidecomplexes.

The first three rounds of selection resulted in a 55-fold increase inphage recovery, based on output/input ratio, and scFvs which only boundto the HLA-A2-EBNA3C complex. Phage display selection results onrecombinant HLA-A2-LLDFVRFMGV and HLA-A2-NLVPMVATV complexes are shownin Table 9.

TABLE 9 Round 1* Round 2* Round 3* Round 4** Input 7.5 × 10¹²  4.9 ×10¹² 2.4 × 10¹²  1.8 × 10¹² Output 4.7 × 10⁶   6 × 10⁶ 8.4 × 10⁷ 1.25 ×10⁸ Output/Input 6.3 × 10⁻⁷ 1.22 × 10⁻⁶ 3.5 × 10⁻⁵  6.9 × 10⁻⁵ FoldEnrichment — 2 55.5 109.5 (From Rd 1) HLA-A2- — — 40/48 (83%) 37/48(77%) EBNA3C Peptide-Specific Clones*** HLA-A2-pp65 — —  0/48 (0%)  0/48(0%) Peptide-Specific Clones*** *Rd 1-3: Panning againstBiotinylated-HLA-A2-pp65 Peptide + Biotinylated-HLA-A2-EBNA Peptide **Rd4: Panning against Biotinylated HLA-A2-EBNA3C Peptide Only ***Relativesignal at least 2-fold greater than background.

These results were somewhat surprising since both of the peptides onHLA-A2 were derived from viral-related proteins, which are not seen inthe human protein repertoire. To confirm these findings, an additionalround of selection was undertaken on just the HLA-A2-EBNA3C complexalone which resulted in a further amplification of recovered phage(109-fold) and a similar percentage of clones which bound to theHLA-A2-EBNA3C complex (83% positive after Round 3 and 77% after Round4).

Bacterial supernatant from individual clones after 3 rounds of phageselection were tested for binding to recombinant,biotinylated-HLA-A2-peptide complexes on vinyl microtiter plates. Whileseveral clones resulted in cross-reactivity to more than just thetargeted HLA-A2-LLDFVRFMGV complex (Clones 335 and 345), Clones 315 and327 were found to have the desired specificity.

Purified EBNA Clone 315 scFv was retested against a similar panel ofrecombinant, biotinylated HLA-A2-peptide complexes. Purified EBNA Clone315 scFv maintained its specificity over a panel of HLA-A2-peptidecomplex in addition to its inability to bind to the native peptide byitself. The anti-HLA-A2 antibody BB7.2 was included to demonstrate thatall HLA-A2-peptide complexes are adherent and presented properly on theplate.

During the screening processes, therefore, several different scFv werefound to bind to the targeted HLA-A2-EBNA3C complex, however only a fewscFv sequences resulted in absolute specificity and did not bind toHLA-A2-peptide complexes of different origins (FIG. 1A). Of those cloneswhich were tested, EBNA Clones 315 and 327 had the same peptide sequenceand were further characterized. After scFv purification, a subsequentvalidation ELISA demonstrates that EBNA Clone 315 maintained itsspecificity towards the targeted HLA-A2-EBNA complex, in addition tofailing to bind to the LLDFVRFMGV peptide by itself (FIG. 1B). Theseinitial binding assays demonstrate the TCR-like binding ability of thisantibody.

Example 18 Affinity Selection of Phage on WT1-Derived RecombinantHLA-A2-Peptide Complex

Antibody selection using phage against biotinylated HLA-A2-RMFPNAPYL(WT1-derived) was done in a similar manner to that which was describedabove. Briefly, the Tomlinson I and J phage display libraries were firstcombined and preincubated with non-biotinylated, irrelevantHLA-A2-NLVPMVATV complex and streptavidin paramagnetic beads.Subsequently, the contents (phage and irrelevant complex) were incubatedwith biotinylated HLA-A2-RMFPNAPYL and captured using fresh streptavidinparamagnetic beads. Once bound to the biotinylated complex, the beadswere washed with PBS containing Tween 20 and the bound phage were elutedfrom the beads using trypsin. After two additional rounds of selection,the recovered phage were used to infect HB2151 E. coli and plated onampicillin-containing agar. The next morning, individual colonies werepicked, cultured overnight in 48-well culture plates, and theirsupernatants tested for the presence of scFv on 96-well ELISA platespre-coated with recombinant HLA-A2-peptide complexes.

The first three rounds of selection resulted in a 90-fold enrichment inphage when comparing the output/input ratios. Phage display selectionresults on recombinant HLA-A2-RMFPNAPYL complex are shown in Table 10.

TABLE 10 Round 1* Round 2** Round 3** Input  3.7 × 10¹² 5.6 × 10¹¹ 1.55× 10¹¹ Output  4.0 × 10⁶ 3.2 × 10⁶ 1.52 × 10⁷ Output/Input 1.08 × 10⁻⁶5.7 × 10⁻⁶  9.8 × 10⁻⁵ Fold Enrichment — 5.3 90.7 (From Rd 1) HLA-A2- —— 3/48 RMFPNAPYL- Specific Clones*** *Rd 1: Panning against 5 μgComplex. **Rd 2-3: Panning against 2.5 μg Complex. ***Relative signal atleast 3-fold greater than background (Irrelevant HLA-A2-Complex).

Bacterial supernatant from three individual clones after three rounds ofphage selection were tested for binding to recombinant,biotinylated-HLA-A2-peptide complexes on vinyl microtiter plates. Allthree clones which were tested had the necessary specificity to onlyrecognize the HLA-A2-RMFPNAPYL complex. It was discovered that all threeclones had the same DNA sequence. Purified WT1 Clone 45 scFv wasretested against a similar panel of recombinant, biotinylatedHLA-A2-peptide complexes. Purified WT1 Clone 45 scFv maintained itsspecificity over a panel of HLA-A2-peptide complex in addition to itsinability to bind to the native peptide outside of the context of MHC.The anti-HLA-A2 antibody BB7.2 was included to demonstrate that allHLA-A2-peptide complexes are adherent and presented properly on theplate.

After screening 48 clones for binding to the specific HLA-A2-RMFPNAPYLcomplex, therefore, three clones were found to bind specifically totheir targeted complex but failed to bind to complexes which displayedan irrelevant peptide (FIG. 2A). Of the clones which were tested, all ofthem were found to have the same peptide sequence and WT1 Clone 45 waschosen for further characterization. After scFv purification, asubsequent validation ELISA demonstrates that WT1 Clone 45 maintainedits specificity towards the targeted HLA-A2-WT1 complex, in addition tofailing to bind to the RMFPNAPYL peptide by itself (FIG. 2B). Theseinitial binding assays demonstrate the TCR-like binding ability of thisantibody.

Example 19 Binding and Specificity Studies with Purified EBNA Clone 315and WT1 Clone 45 scFvs on Peptide-Pulsed T2 Cells

To demonstrate that the isolated EBNA Clone 315 and WT1 Clone 45 scFvsare able to recognize and bind to their native complexes on the surfaceof peptide-pulsed antigen presenting cells (APCs), the TAP-deficient T2cell line was used. T2 cells were first incubated for 5 hours at 37° C.with either LLDFVRFMGV (EBNA3C-derived), RMFPNAPYL (WT1-derived) orirrelevant peptide KLQCVDLHV in serum-free medium containing β₂M. Thecells were subsequently washed and stained with the purified WT1 Clone45, EBNA Clone 315 or an irrelevant scFv. In addition, peptide pulsedand unpulsed T2 cells were also incubated with an anti-HLA-A2-FITC(BB7.2) antibody. This BB7.2 staining control was included due toprevious studies which demonstrate that if a peptide is able to bindHLA-A2 on the T2 cell surface, the HLA-A2 molecule is stabilized, andthe stabilization can be visualized by an increase in fluorescenceintensity (81). As shown in FIG. 3A, T2 cells which have been pulsedwith either the LLDFVRFMGV or KLQCVDLHV peptides resulted in afluorescence shift, consistent with their binding to the HLA-A2 pocket.However, EBNA Clone 315 was only able to stain T2 cells pulsed with itsspecific target peptide LLDFVRFMGV and not an irrelevant peptide (FIG.3B). Similar results were obtained when T2 cells were pulsed with eitherthe RMFPNAPYL or LLDFVRFMGV peptides and stained with WT1 Clone 45 scFv.While both peptides were able to stabilize the HLA-A2 molecule (FIG.4A), WT1 Clone 45 scFv was only able to detect the T2 cells pulsed withthe RMFPNAPYL peptide (FIG. 4B). This further validates their utility indetecting the native complex on the surface of cells.

Next, the detection sensitivity of the EBNA Clone 315 scFv using flowcytometry was evaluated in order to correlate sensitivity with antigendensity using flow cytometric quantitative beads. Briefly, TAP-deficientT2 cells were pulsed with (solid, unfilled lines) or without (dashed,unfilled lines) LLDFVRFMGV (FIG. 3A, top, left panel) or KLQCVDLHVpeptides (FIG. 3A top, right panel) at 20 μM in serum-free IMDM media at37° C. for 5 hours. The cells were then stained with a mouse-anti-humanHLA-A2-FITC conjugated antibody (unfilled lines) or a control mouseIgG₁-FITC conjugated antibody (filled lines) and analyzed on the FACSmachine. Peptide-pulsed T2 cells from A were stained with EBNA Clone 315scFv (unfilled lines) or a control scFv (filled lines) (FIG. 3B). OnlyT2 cells which had been pulsed with the LLDFVRFMGV peptide (left panel),but not ones which had been pulsed with KLQCVDLHV (right panel), couldbe stained by the EBNA Clone 315 scFv (FIG. 3B). T2 cells were incubatedwith decreasing concentrations of the LLDFVRFMGV peptide andsubsequently stained with EBNA Clone 315 scFv as above (FIG. 3C). Basedon geometric mean fluorescence (control scFv background subtracted), thelower limit of detection corresponds with 78 nM of peptide used forpulsing.

By titrating down the amount of peptide used for incubation with the T2cells, it was determined that concentrations as low as 78 nM were stillable to produce a fluorescence signal above background when stained withEBNA Clone 315 scFv (FIG. 3C). With decreasing concentrations of peptideused for loading, there was a corresponding reduction in overall HLA-A2intensity (data not shown) as one would expect.

Similarly, FIG. 4 shows that WT1 Clone 45 can recognize HLA-A2-RMFPNAPYLon peptide-pulsed T2 cells. TAP-deficient T2 cells were pulsed with(solid, unfilled lines) or without (dashed, unfilled lines) RMFPNAPYL(left panel) or LLDFVRFMGV peptides (right panel) at 40 μM in serum-freeIMDM media at 37° C. for 5 hours. The cells were then stained with amouse-anti-human HLA-A2-FITC conjugated antibody (unfilled lines) or acontrol mouse IgG1-FITC conjugated antibody (filled lines) and analyzedon the FACS machine (FIG. 4A). Peptide-pulsed T2 cells from A werestained with WT1 Clone 45 (unfilled lines) or a control scFv (filledlines). Only T2 cells which had been pulsed with the RMFPNAPYL peptide(left panel), but not ones which had been pulsed with LLDFVRFMGV (rightpanel), could be stained by the WT1 Clone 45 (FIG. 4B).

Example 20 Demonstrating HLA Restriction of the LLDFVRFMGV Peptide andEBNA Clone 315 Using Peptide-Pulsed BLCLs

The expression of these peptides on BLCLs, especially since BLCLs areused routinely as APCs (82), was examined. Two BLCL lines were used, oneHLA-A2⁺ (DIMT) and one HLA-A2⁻ (6268A) (FIG. 5A). The BLCLs wereincubated in serum-free IMDM media for 5 hours at 37° C. with either thespecific LLDFVRFMGV or irrelevant KLQCVDLHV peptides. When incubatedwith the specific peptide, only the HLA-A2⁺ DIMT BLCL could be stainedby EBNA Clone 315 (FIG. 5B). Similarly to results seen with T2 cells,DIMT cells loaded with the irrelevant peptide, or 6268A loaded with thespecific/irrelevant peptide, could not be stained with EBNA Clone 315.It is interesting to note that without peptide pulsing we wereunsuccessful at staining DIMT. While our staining approach has beenoptimized to detect low levels of antigen through signal amplificationinvolving secondary and tertiary reagents to detect the scFv, the amountof peptide that the cell naturally presents seems to be below our levelof detection.

Subsequently, in an attempt to study the duration of peptidepresentation on HLA-A2, a pulse-chase experiment was set up to monitorthe levels of the HLA-A2-EBNA3C complex on DIMT cells over time.Initially, DIMT cells were incubated in serum-free IMDM media for 5hours at 37° C. with the LLDFVRFMGV peptide. Afterwards, the cells werewashed twice with RPMI+10% FBS and further cultured in this media for anadditional 5 hours and 24 hours. At each of these three time points,cells were harvested and stained with either the purified EBNA Clone 315scFv or an irrelevant scFv. The results show that after pulsing theHLA-A2-EBNA3C complex could easily be detected on the cell surface (FIG.5C). Interestingly, even after the cells were transferred to fresh mediaand cultured for an additional 5 and 24 hours, the MHC-peptide complexcould still be detected, signifying that peptide-pulsed BLCLs are ableto hold onto and present antigen for at least a day after the peptidehad been removed from the media. This data further supports the use ofautologous BLCLs in the generation of antigen specific T cells and theutility of TCE-specific antibodies like EBNA Clone 315 in precisevisualization of TCE expression on APCs or target cells.

Example 21 Construction of EBNA Clone 315 and WT1 Clone 45 scFv-FcFusion Proteins

Initially, the scFv sequences were made compatible for cloning into ascFv-Fc expression vector by using PCR to add the desired restrictionenzyme sites (NheI and ApaI) to either side of the EBNA Clone 315 andWT1 Clone 45 scFv sequences. The PCR reaction was done on the Tomlinsonlibrary vector which contained the WT1 Clone 45 and EBNA Clone 315 scFvsequences (FIG. 7A). After subsequent digestion using NheI and ApaI, thedigested PCR products were removed from a 1% agarose gel and purified(FIG. 6B).

With regards to cloning and expression of the scFv-Fc fusion proteins, aproprietary vector obtained from Eureka therapeutics(IgG Vector) wasused. The first constant heavy chain (CH₁) was removed from this vector,something which is typically done when generating Fc fusion proteins(83). Once generated, the vector was digested with NheI and ApaI andthen ligated to the predigested PCR products from FIG. 7B. The ligatedproducts yielded a vector which expressed the EBNA Clone 315 or WT1Clone 45 scFv genes in tandem to the CH_(2,3) domains of a human IgG1under a single CMV promoter (scFv-Fc Vector; FIG. 7B). After furthervalidation using DNA sequencing, the two fusion constructs werelinearized using HindIII and ran on a 1% agarose gel. Digestion withHindIII also allowed us to block the expression of the light chain thatis still present in the vector, which for all intensive purposes wasundesired. As expected, both digested plasmids ran at the anticipatedsize (˜11,000 bp) based on their location relative to the lambda HindIIImarker. Each linearized plasmid was subsequently introduced into DG44cells and cultured in OptiCHO media as described in Example 10 above.

Example 22 Binding Kinetics and Sensitivity of EBNA Clone 315 scFv-Fc onRecombinant HLA-A2-Peptide Complex and Peptide-Pulsed T2 Cells

To further understand the affinity of the interaction between EBNA Clone315 and the HLA-A2-EBNA3C complex, surface plasmon resonance was used todetermine the binding kinetics between these two proteins. First, theEBNA Clone 315 scFv-Fc was purified and its binding ability was testedusing ELISA (FIG. 8A) along with flow cytometry via peptide-pulsed T2cells at varying concentrations (FIGS. 8B and C). These initial studiesdemonstrate that the antibody maintains its binding characteristics whenexpressed as a fusion protein In addition, it is important to note thatthe flow cytometric sensitivity of the scFv and scFv-Fc were verycomparable (200 nM-20 nM), further highlighting the utility of the scFvas a monomeric binding fragment.

Next, using the Biacore T100 (GE Healthcare), a CM5 chip (flow cells 1and 2) was initially activated for amine coupling based on manufacturerrecommendation. The purified EBNA Clone 315 scFv-Fc was subsequentlyimmobilized onto the second flow cell and the purified HLA-A2-EBNA3Ccomplex passed over both flow cells as part of the soluble phase. Afterbackground subtraction (signal from flow cell 2 minus that of flow cell1), the association rate (k_(on)) and dissociation rate (k_(off)) weredetermined (2.361×10⁵ M⁻¹s⁻¹ and 6.891×10⁻²s⁻¹, respectively), resultingin an overall K_(D) (k_(off)/k_(on)) of 291 nM using a 1:1 binding model(FIG. 9); these kinetic rates were very similar to previously isolatedFabs against different MHC-peptide complexes (22, 31). Relative topublished TCR:MHC Class I-peptide K_(D) measurements, which typicallyrange in the neighborhood of 2-50 μM (84), our scFv:MHC Class I-peptideinteraction seems to be at best 150-fold stronger, with the mostsignificant improvement attributed to a slower k_(off). Previous studieswhich support an affinity-based T cell activation model argue that agreater overall affinity or slower dissociation rate leads to higherinterferon-gamma release and target cell lysis (85, 86).

Lastly, in an attempt to quantify the amount of HLA-A2-LLDFVRFMGVcomplex on the surface of peptide-pulsed T2 cells, we decided to useflow cytometric quantitation beads. This data will also be useful indetermining the detection threshold of EBNA Clone 315 scFv and scFv-Fc.First, purified EBNA Clone 315 scFv-Fc was conjugated to a fluorescentlabel, Alexa Fluor 647 using a commercially available kit. Subsequently,T2 cells were pulsed with 20, 10, 5, or 0 μM of LLDFVRFMGV peptide at37° C. for 5 hours. After pulsing, the peptide-pulsed T2 cells, alongwith beads containing known quantities of anti-human IgG₁ antibodies,were incubated with the fluorescently-labeled EBNA Clone 315 scFv-Fc.Once the cells and beads were analyzed on the FACS machine, thefluorescence intensities were correlated to each other, resulting in anestimation of the number of complexes on the surface of the T2 cellsrelative to the quantity of peptide used for pulsing. These four values(337,091 sites with 20 μM, 149,688 sites with 10 μM, 76,040 sites with 5μM, and no sites with 0 μM) were plotted on a graph and a trendline wasused to create a standard curve (R²=0.9948) (FIG. 10A). Furthermore,when looking at the lower end of the spectrum, we have determined thatan amount less than 40 nM of peptide will correspond to less than 100complexes on the surface of the cell (FIG. 10B), placing the detectionlevel of the EBNA Clone 315 scFv-Fc fusion within that range.

Example 23 Binding and Specificity Studies of WT1 Clone 45 scFv-Fc onRecombinant HLA-A2-Peptide Complex and Peptide-Pulsed Cells

In order to do further studies regarding the presentation of theRMFPNAPYL peptide on the surface of APCs, the WT1 Clone 45 scFv-Fcfusion protein was first purified and validated for binding to itstargeted recombinant HLA-A2-peptide complex (FIG. 11A). As was shownwith the scFv, the fusion protein maintained its binding ability on theELISA plate. Next, we decided to check and see if the scFv-Fc maintainedits binding ability and specificity on peptide-pulsed T2 cells. T2 cellswere pulsed with the RMFPNAPYL peptide or an irrelevant peptide inserum-free media with β₂M at 37° C. for 5 hours. Using flow cytometry,the scFv-Fc was able to detect T2 cells which had been pulsed with theRMFPNAPYL peptide, but failed to recognize the cells pulsed with anirrelevant peptide (FIG. 11B). These two assays further validated thatthe fusion protein acts in the same way as the original scFv.

Subsequently, we decided to test whether the binding of the RMFPNAPYLpeptide and scFv-Fc fusion protein were restricted to HLA-A2. HLA-A2⁺and HLA-A2⁻ BLCLs (DIMT and 6268A, respectively) were pulsed with theRMFPNAPYL peptide in serum-free media at 37° C. for 5 hours. Similarlyto EBNA Clone 315, the WT1 Clone 45 scFv-Fc fusion protein was only ableto recognize the peptide pulsed DIMT and not the HLA-A2⁻ 6268A BLCL(FIG. 12). These results demonstrate that the RMFPNAPYL peptide isrestricted to HLA-A2 and WT1 Clone 45 is only able to recognize it inthe context of this complex.

Example 24 Antibody-Dependent Cellular Cytotoxicity (ADCC) of EBNA Clone315 scFv-Fc on Peptide-Pulsed Cells

In addition to using the scFv-Fc for antigen presentation studies, wetested whether the truncated human IgG₁ Fc region is capable of inducingantibody-dependent cellular cytotoxicity (ADCC). In order to avoidvariability amongst human donor lymphocytes, and in an effort toincrease the chances of observing cytotoxicity, Hong-fen Guo in ourlaboratory generated a CD16(V)-transduced NK92MI cell line. This NK92cell variant is transduced with both IL-2 and the human CD16 activatingFc receptor (FcγRIIIA) containing a high affinity polymorphism (valineinstead of phenylalanine at position 158 on CD16) responsible for anenhancement in ADCC and clinical response to antibody-basedimmunotherapy (87, 88).

We used this cell line in combination with the EBNA Clone 315 scFv-Fc oran irrelevant, isotype-matched scFv-Fc to test whether the fusionprotein can induce NK92MI-mediated ADCC against LLDFVRFMGV-pulsed LUY(HLA-A2⁺) BLCL. At an E:T ratio of 42:1, EBNA Clone 315 scFv-Fc led togreater killing over background (with or without an irrelevant scFv-Fc)at the two highest concentrations tested (27-32% versus 13-15%) (FIG.13). A similar magnitude of killing (over background) was also observedwith other peptide-pulsed, HLA-A2⁺ target BLCLs (DIMT and JG19). Theseresults show that these truncated scFv-Fc fusion proteins maintain theirFc-mediated effector functions, despite being about 33% smaller than afull immunoglobulin.

Example 25 Construction and Retroviral Transduction of anHLA-A2-RMFPNAPYL-Specific Chimeric Antigen Receptor into NK92MI Cells

In order to generate a CAR specific for the HLA-A2-RMFPNAPYL complex,the WT1 Clone 45 scFv would typically be fused to intracellularsignaling domains of immune-modulatory proteins found in immune effectorcells. A CAR expression vector (St. Jude CAR) in which a CD19-specificscFv is fused to the CD8α hinge/transmembrane region, 4-1BB and CD3ζchain was obtained and modified so that the anti-CD19 scFv was replacedwith a WT1 Clone 45 scFv. However, due to restriction enzymeincompatibility issues between the St. Jude CAR vector and the Tomlinsonlibrary vector used for PCR, the entire CAR gene, containing the WT1Clone 45 scFv, was commercially synthesized by Genescript. The resultingWT1 pUC57 vector contained the desired WT1 Clone 45 CAR sequence flankedby EcoRI and XhoI.

An additional feature to the St. Jude CAR vector is an IRES-GFP sequencedownstream of the CAR sequence. This allows for direct correlation ofCAR expression with GFP without having to fuse both proteins together.In order to take advantage of this feature, we digested the WT1 pUC57vector and St. Jude CAR vector using EcoRI and XhoI. Afterwards, thedigested and undigested plasmids were run on a 1% agarose gel along withthe lambda HindIII and 100 bp markers. The highlighted bandscorresponded to the anticipated sizes of the St. Jude plasmid lackingthe CAR sequence (˜6500 bp) and the WT1 Clone 45 CAR sequence lackingthe pUC57 plasmid (˜1500 bp). These bands were excised from the gel, andafter DNA purification, the two were ligated together. After theligation products were transformed into E. coli, 8 colonies wereselected at random and their plasmids isolated. The isolated plasmidswere then validated by sequencing to determine whether they contain theWT1 Clone 45 scFv sequence in the context of the CAR. In addition, theplasmids were also digested with EcoRI and XhoI and run on a 1% agarosegel along with lambda HindIII and 100 bp markers to validate theirsizes. After demonstrating that both bands from each plasmid yielded theexpected sizes, it was determined that the cloning was successful.

Once the WT1 Clone 45 CAR was generated (FIG. 23), the DNA was packagedinto retrovirus using the 293T-based GP2 cell line. Once the retroviruswas generated in the culture media, it was recovered and concentrated.The concentrated virus was then used to infect 500,000 to 1,000,000NK92MI cells in NK92MI growth media. After 3-4 days of culture, theNK92MI cells infected with the retrovirus were compared to mock-infectedcells (infected with empty retrovirus) with regards to GFP expressionusing flow cytometry. While the infection efficiency was approximately27.5%, flow assisted cytometric cell sorting (FACS) allowed us to enrichthe GFP-positive population to more than 98% positive (FIG. 23).

Example 26 Construction and Retro Viral Transduction of anHLA-A2-LLDFVRFMGV-Specific Chimeric Antigen Receptor into NK92MI Cells

Since the WT1 Clone 45 CAR required us to purchase the WT1 pUC57 vector,additional restriction sites were added to this construct for greaterease when swapping different scFvs. As a result, the EBNA Clone 315 scFvsequence could directly be cloned out of the Tomlinson vector from whichit was derived.

The first cloning step involved the removal of the WT1 Clone 45 scFvfrom the WT1 pUC57 vector using SfiI and NotI. The same digestion wasdone to the Tomlinson vector containing the EBNA Clone 315 scFvsequence. Once digested, both plasmids were run on a 1% agarose gelalong with lambda HindIII and 100 bp markers. The highlighted bandscorresponded to the WT1 pUC57 vector without a scFv, and the EBNA Clone315 scFv excised from the Tomlinson vector. These bands were excisedfrom the gel, the DNA was purified, and ligated together to yield theEBNA pUC57 vector. The ligation products were subsequently transformedinto E. coli, 10 colonies were selected at random, their plasmids werepurified, and each DNA digested with EcoRI alone or EcoRI and XhoI. Asanticipated, due to an inherent XhoI site within every scFv sequencederived from the Tomlison vector (with the exception of WT1 Clone 45scFv in the context of pUC57 since the site was removed when purchasedas a CAR from Genescript), the double digestion yielded three separatebands.

For the second cloning step, in which the EBNA Clone 315 CAR sequencewas excised from the pUC57 vector and added to the St. Jude CAR vector,a partial digestion of the EBNA pUC57 vector using XhoI was necessary.The EBNA pUC57 plasmids isolated from the 10 colonies above werecombined and digested with XhoI at room temperature for 1 minute. Thereaction was quickly stopped by adding it to 4 separate wells of a 1%agarose gel and running the DNA along with uncut plasmid, lambda HindIIIand 100 bp markers. The highlighted bands were determined to be theexpected size of the linearized EBNA pUC57 plasmid (˜4300 bp); thislinearized plasmid is a result of a random cut at either of the two XhoIsites. Subsequently, to obtain the complete CAR sequence (˜1500 bp), thelinearized plasmid was isolated from the gel and digested completelywith EcoRI. The resulting double and single digests were run on a 1%agarose gel along with the lambda HindIII and 100 bp markers. Thehighlighted band corresponded to the anticipated size of the EBNA Clone315 CAR gene, and as a result was excised from the gel, DNA purified,and ligated to the predigested (EcoRI and XhoI) St. Jude CAR vector.After the ligation products were transformed into E. coli, 10 colonieswere selected at random and their plasmids isolated. The isolatedplasmids were then validated by sequencing to determine whether theycontain the EBNA Clone 315 scFv sequence in the context of the CAR. Inaddition, the plasmids were also digested with EcoRI and run on a 1%agarose gel along with the lambda HindIII marker to validate theirsizes. After demonstrating that the bands yielded the expected sizes, itwas determined that the cloning was successful.

After the EBNA Clone 315 CAR was generated (FIG. 14), the DNA waspackaged into retrovirus and used to infect NK92MI cells in the same wayas with the WT1 Clone 45 CAR. After 3-4 days of culture, the GFPexpression level of the infected NK92MI cells were compared tomock-infected cells. The initial infection efficiency was approximately24%, and after flow assisted cytometric cell sorting (FACS), theGFP-positive population was enriched to more than 90% positive (FIG.14).

Example 27 EBNA Clone 315 CAR-Equipped NK92MI Cells can Detect CellsBearing the Specific HLA-A2-LLDFVRFMGV Complex via CD107a Expression

Once the NK92MI cells were enriched for EBNA Clone 315 CAR expression,they were tested for their ability to recognize the targetedHLA-A2-EBNA3C complex. As an initial readout of NK92MI activation bytarget cells, we assayed for cell surface expression of CD107a, a markerof NK cell and T cell degranulation (90, 91). T2 cells were loaded with20 μM of the targeted peptide (LLDFVRFMGV), an irrelevant peptide(YMFPNAPYL) or no peptide. Using a 1:1 E:T ratio, the T2 cells werecocultured with EBNA Clone 315 CAR-expressing NK92MI cells in thepresence of an anti-CD107a-PE conjugated antibody at 37° C. for 5 hours.As shown in FIG. 16A, NK92MI cells equipped with the EBNA Clone 315 CARdid not react to unpulsed T2 cells or T2 cells pulsed with theirrelevant peptide, showing CD107a levels comparable to those of NK92MIcells cultured in the absence of targets. On the other hand, when theCAR-equipped NK92MI cells were cocultured with T2 cells that had beenpulsed with the LLDFVRFMGV peptide, 27% of GFP⁺ cells expressed CD107aabove background levels. These results show that after scFv engineering,the CAR is able to maintain its specificity towards the targetedHLA-A2-peptide complex.

Next, in order to get a quantitative measurement of how sensitive thisCAR is at activating NK92MI cells, we titrated down the LLDFVRFMGVpeptide concentration used to pulse T2 cells and measured their abilityto activate the CAR-equipped NK92MI cells. As can be seen in FIG. 16B,the lower limit of response by the CAR-equipped NK92MI was at a peptideconcentration of 10 nM, with a clear dose response curve beginning atthe 600 nM concentration. Based on our earlier quantitation studies,this peptide concentration corresponds to approximately 25 complexes onthe cell surface. Compared to the levels necessary for epitope detectionusing the EBNA Clone 315 scFv or scFv-Fc (200-20 nM), the CAR seems tobe a more sensitive approach at detecting low levels of MHC-peptidecomplex on the surface of APCs using flow cytometric analysis.

While T2 cells can present any peptide of interest, BLCLs naturallypresent their own peptides on their MHC Class I molecules. Similarly toT2, these endogenous peptides can be replaced by simple incubation witha substitute peptide of high enough affinity. Using a 1:1 E:T ratio,HLA-A2⁺ (DIMT) and HLA-A2⁻ (6268A) BLCLs were pulsed with serum-freeIMDM medium or medium containing the LLDFVRFMGV, cocultured with EBNAClone 315 CAR-expressing NK92MI cells as discussed above, and assayedfor CD107a expression using flow cytometry. Peptide pulsed DIMT(HLA-A2⁺) induced 25% of GFP⁺ NK92MI cells to express CD107a (FIG. 17),in contrast to 0.54% for peptide pulsed 6268A (HLA-A2⁻) and 1.09% forunpulsed DIMT. This data further demonstrates both peptide specificityand HLA-A2 exclusivity of the EBNA Clone 315 CAR.

Example 28 EBNA Clone 315 CAR-Equipped NK92MI Cells can Destroy CellsBearing the Specific HLA-A2-LLDFVRFMGV Complex via ⁵¹Cr Release

While CD107a expression on NK cells and T cells reflect theiractivation, target cell lysis can also be measured using a conventional⁵¹Cr cytotoxicity assay. First, to get an idea of how sensitive the ⁵¹Crcytotoxicity assay is with regards to killing HLA-A2-EBNA3C expressingtargets, T2 cells were pulsed with decreasing concentrations of theLLDFVRFMGV peptide at 37° C. for 3 hours and subsequently labeled with⁵¹Cr as described in the Materials and Methods. The labeled T2 cellswere then cocultured with EBNA Clone 315 CAR-expressing NK92MI cells at37° C. for 2 hours at a 3:1 E:T ratio. Similar to the results seen inthe CD107a assay (FIG. 16B), EBNA Clone 315 CAR expressing NK92MI cellswere able kill T2 cells in a peptide-dependent manner, with 13.2% of 2nM peptide-pulsed T2 cells being killed compared to 10.1% with unpulsedT2 cells (FIG. 18). Relative to that which can be detected using flowcytometric antibody staining, the level of sensitivity is in the orderof 10-100 fold greater in favor of the CAR using two separate assays(CD107a and ⁵¹Cr).

Next, DIMT and 6268A BLCLs pulsed with the LLDFVRFMGV peptide (20 μM) inserum-free IMDM (FIG. 19A and B) were used as targets in the ⁵¹Crrelease assay. Similar to the results from the CD107a assay (FIG. 17),only the HLA-A2⁺ DIMT BLCL were lysed by the CAR-equipped NK92MI cells(FIG. 19A), which could be blocked using the purified EBNA Clone 315scFv-Fc (FIG. 19B). In addition, the ability to block cytotoxicity wasnot restricted to the scFv-Fc protein since a commercial anti-HLA-A2(BB7.2) antibody also possessed blocking ability (data not shown). Thisblocking data recapitulates results seen with other MHC-restricted,peptide-specific antibodies on antigen-specific cytolytic T cells.

Although the lytic potential of the CAR-equipped NK92MI cells wasclearly evident when targets were artificially pulsed with the relevantpeptide, cytotoxicity against naturally processed HLA-A2-peptidecomplexes is of clinical relevance. Here, CAR-equipped NK92MI cells weretested against a panel of HLA-A2⁺ (DIMT and JG19) and HLA-A2⁻ (6268A andGKO) unpulsed BLCLs. While the level of cytoxicity was low, 23.0% forDIMT and 8.9% for JG19 (30:1 E:T ratio), this was highly significantwhen compared to 3.6% for GKO and 1.8% for 6268A (FIG. 20A). Inaddition, when the cytotoxicity assay was performed in the presence ofEBNA Clone 315 scFv-Fc, the killing capacity could be reduced byapproximately 46% when compared to that with an irrelevant scFv-Fc or inthe absence of antibody (FIG. 20B). These findings demonstrate theutility and specificity of TCR-like CARs in reprogramming effectorimmune cells to engage antigen whose expression is below the detectionlimit using conventional flow cytometry.

Lastly, since both the EBNA Clone 315 CAR and scFv-Fc fusion proteinhave the same variable sequences used for detecting theHLA-A2-LLDFVRFMGV complex, we decided to directly compare CAR-mediatedcytotoxicity with ADCC since both approaches are currently being usedindependently for the treatment of cancer patients. First, the DIMT BLCLwas pulsed with the LLDFVRFMGV peptide at 20 μM in serum-free IMDM mediaat 37° C. for 2 hours. The pulsed BLCL was then labeled with ⁵¹Cr andcocultured with either EBNA Clone 315 CAR or CD16(V)-expressing NK92MIcells along with EBNA Clone 315 scFv-Fc or an irrelevant scFv-Fc at anE:T ratio of 15:1 for 3 hours at 37° C. At a EBNA Clone 315 scFv-Fcconcentration of 0.5 μg/ml, CD16(V) NK92MI cells were able to kill about30-35% of cells, compared to 10-15% with an irrelevant scFv-Fc or noantibody at all (FIG. 21). When the ADCC experiment was carried outusing higher scFv-Fc concentrations, the cytotoxicity percentage did notchange (data not shown). On the other hand, at the same E:T ratio, EBNAClone 315 CAR-equipped NK92MI cells were able to kill 80-90% of the samepeptide-pulsed target cells; and the EBNA Clone 315 scFv-Fc was includedas a blocking control (FIG. 22). These results demonstrate that theCAR-mediated killing involving NK92MI cells is a far more potent meansof target cell lysis compared to ADCC in our setting.

Example 29 WT1 Clone 45 CAR-Equipped NK92MI Cells can Destroy CellsBearing the Specific HLA-A2-RMFPNAPYL Complex via ⁵¹Cr Release

Along with the EBNA Clone 315 CAR, we decided to test the cytolyticability of the WT1 Clone 45 CAR in the context of NK92MI cells. First,DIMT and 6268A BLCLs were pulsed with the RMFPNAPYL peptide (40 μg/ml)in serum-free IMDM at 37° C. for 3-5 hours. Subsequently, the targetcells were labeled with ⁵¹Cr and cocultured with the CAR-equipped NK92MIcells at 37° C. for 4 hours. Of the two peptide-pulsed BLCLs, only theHLA-A2⁺ DIMT could be lysed (˜70% versus ˜5% with 6268A) at a 10:1 E:Tratio (FIG. 24). In addition, CAR-mediated cytotoxicity could be blockedusing a commercial anti-HLA-A2 antibody by approximately 45% (FIG. 25),further demonstrating specificity.

Next, we decided to tested the cytolytic capacity of WT1 Clone 45CAR-equipped NK92MI cells against cell lines which might nativelyexpress the HLA-A2-RMFPNAPYL complex. Due to previously published data(92), and conversations with Dr. Richard O'Reilly's laboratory here atMSKCC, researchers have demonstrated that WT1 can be constitutivelyactivated in all BLCLs derived from EBV immortalization. Morespecifically, O'Reilly's group was able to show WT1 transcript in theDIMT BLCL (data not shown). As a result, we first decided to test WT1Clone 45 CAR-mediated killing against unpulsed HLA-A2⁺ DIMT and HLA-A2⁻6268A BLCL. Similarly to what was seen with the EBNA Clone 315 CAR, WT1Clone 45 CAR-equipped NK92MI cells were able to kill unpulsed DIMT at alower capacity than peptide-pulsed DIMT. While the level of cytotoxicitywas lower, ˜35% for DIMT at a 20:1 E:T ratio, it was far greater whencompared to 6268A (˜5%) (FIG. 26A). In addition, when the cytotoxicityassay was performed in the presence of the WT1 Clone 45 scFv-Fc, thekilling capacity could be reduced by approximately 43% relative to anirrelevant scFv-Fc or in the absence of antibody (FIG. 26B). Thesefindings correspond well with what was seen using the EBNA Clone 315 CARand further demonstrate the utility and specificity of TCR-like CARs inreprogramming effector immune cells to engage antigen.

Lastly, CAR-mediated cytotoxicity against two cell lines which areHLA-A2-positive and previously shown to express WT1 was tested. OVCAR-3is a cell line established from malignant ascites of a patient withprogressive adenocarcinoma of the ovary (93) and later shown to containWT1 mRNA (94). In addition, 697 is a human pre-B cell leukemiaestablished from bone marrow cells obtained from a child with relapsedacute lymphocytic leukemia (ALL) (95). Since then, several groups haveshown that this cell line also expresses high levels of both WT1transcript and protein (96, 97). WT1 Clone 45 CAR-expressing NK92MIcells were cocultured with ⁵¹Cr labeled OVCAR-3 and 697 cells at 37° C.for 4 hours. CAR-equipped NK92MI cells were able to lyse approximately20-30% of 697 and OVCAR-3 cells at a 20:1 E:T ratio, which decreasedwith the number of effector cells used in the assay. This datademonstrates that these two cell types are sensitive to WT1 Clone 45CAR-equipped NK92MI cells and provides further evidence for theirutility in the treatment of HLA-A2⁺/WT1 + malignancies.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and Examples detail certain embodiments of the invention anddescribes the best mode contemplated by the inventors. It will beappreciated, however, that no matter how detailed the foregoing mayappear, the invention may be practiced in many ways and the inventionshould be construed in accordance with the appended claims and anyequivalents thereof.

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1. (canceled)
 2. The isolated antigen-binding protein of claim 6,wherein the isolated antigen-binding protein is an antibody.
 3. Theisolated antigen-binding protein of claim 6, wherein the antigen-bindingprotein is a chimeric antigen receptor (CAR).
 4. The isolatedantigen-binding protein of claim 2, wherein the antibody is afull-length antibody, a substantially intact antibody, a Fab fragment, aF(ab′)₂ fragment or a single chain variable fragment (scFv).
 5. Theisolated antigen-binding protein of claim 6, wherein the antigen-bindingprotein specifically binds to an epitope on an HLA/peptide complex. 6.The isolated antigen-binding protein of claim 5, comprising one of: (A)an antigen binding region having the amino acid sequence of SEQ ID NO:2; (B) an antigen binding region comprising a V_(H) and a V_(L)respectively, with the amino acid sequences SEQ ID NOs: 22 and 23; or(C))(i) (a) a light chain(LC) complementarity determining region 1(CDR1) comprising the amino acid sequence of SEQ ID NO: 56; (b) a LCCDR2 comprising the amino acid sequence of SEQ ID NO: 57; and (c) a LCCDR3 comprising the amino acid sequence of SEQ ID NO: 64; and (ii) (a) aheavy chain (HC) complementarity determining region 1 (CDR1) comprisingthe amino acid sequence of SEQ ID NO: 38; (b) a HC CDR2 comprising theamino acid sequence of SEQ ID NO: 40; and (c) a HC CDR3 comprising theamino acid sequence of SEQ ID NO:
 48. 7. The isolated antigen-bindingprotein of claim 6, wherein the peptide of the HLA /peptide complex hasthe amino acid sequence RMFPNAPYL (SEQ ID NO:1). 8.-18. (canceled) 19.The isolated antigen-binding protein of claim 6, wherein the HLA of theHLA/peptide complex is HLA-A2.
 20. A fusion protein comprising anantigen-binding protein of claim
 6. 21. An isolated single-chainvariable fragment (scFv) comprising one of the amino acid sequences setforth in SEQ ID NOS:
 2. 22. An isolated scFv comprising a V_(H) and aV_(L) linked by an amino acid spacer, wherein the V_(H) and V_(L)respectively comprise the amino acid sequences set forth in SEQ ID NOS:(i) 22 and
 23. 23. An immunoconjugate comprising a first component whichis an antigen-binding protein or fragment thereof of claim
 6. 24. Theimmunoconjugate of claim 23, comprising a second component having asecond amino acid sequence.
 25. The immunoconjugate according to claim24, further comprising a cytotoxin.
 26. The immunoconjugate of claim 24,wherein the second component is a binding protein or antibody having abinding specificity for a target that is different from the HLA-peptidecomplex.
 27. A bispecific antibody comprising an antigen-binding proteinof claim
 6. 28. A pharmaceutical composition comprising a compound ofclaim 6.